Early in development, Portal 2 was planned to feature no portals at all. Valve focused on a new gameplay mechanic called "F-Stop", and though the new mechanic received some positive feedback, every single play tester was disappointed with the absence of portals. Valve then revised the game to include portals.

There are unused audio files in Portal 2 that suggest there was once a scene where Caroline was forcibly transformed into GLaDOS.

'No listen to me! Sir, I do not want this.'
'I do not want this!'
'Mr. Johnson, I don't want this!'
'Cave, I'm begging you, don't! Please!'
'NOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO!'
*GLaDOS voice* 'I am awake. And I am going to kill you all.'

During development, Cave Johnson's "secretary" was a scientist named Greg. But, Valve realized that they would have to hire another voice actor. In order to cut costs and enticed to create an origin for GLaDOS, Greg was cut and Caroline was added. Greg was later used as Cave Johnson's new partner in the Perpetual Testing Initiative DLC.

In the Spanish version of Portal 2, at the end of chapter 5, when Wheatley takes over GLaDOS' body, Wheatley tries to show his intelligence to Chell by using the translator and in doing so, winds up saying, "It seems that you are using this translation too incorrectly. Please check the manual."

Because of the failure of the attempted meme of Hoopy the Hoop in Portal, VALVe included the model of Hoopy the Hoop in 3 spots in Portal 2: Near the deactivated GLaDOS in Chapter 1, In Chapter 2 Chamber 2, and behind a desk in a Ratman Den.

During the development of the game, the team stated that there would be no cake jokes, as they were tired of the "The Cake Is A Lie" meme. Eventually, it turns out that they lied about this, as they "couldn't resist putting in just one."

During the "Bring Your Daughter to Work Day" science-fair, you will find a giant potato that is growing to the ceiling. If you look around the potato, you will find that it was made by Chell and she used a "special ingredient from daddy's work." This indicates that Chell was the daughter of an Aperture Science employee.

In one of Ratman's rooms is a radio blaring strange noises. This "noise" is actually a SSTV signal. If you decode it, it will show an image from the end of the game.

In chapter 5, there is a turret that you can rescue for being "different." This turret (named the Oracle Turret by fans and programers) will spout off some several cryptic messages that relate to the later in the game:
• "Get mad. Don't make lemonade." - This is a quote from Cave Johnson.
• Spoiler:"Prometheus was punished by the gods for giving the gift of knowledge to man. He was cast into the bowels of the earth and pecked by birds." - This is referencing how Wheatly is given all of GLaDOS's power and turns GLaDOS into a potato, throws her and you into the underground labs, and GLaDOS is pecked at by a bird.
• Spoiler:"It won't be enough." - This reveals that the paradox will not work on Wheatly.
• Spoiler:"The answer is beneath us." - This refers to the underground labs.
• Spoiler:"Her name is Caroline." - Caroline being GLaDOS's real name.

An unused soundfile for the Announcer exists in Portal 2.
He says: "We will attempt to comply with these now non-existent agencies by playing some more smooth jazz."
No smooth jazz will play after that.

However, in the German version, the music can be heard.

The original idea for the game was to replace Chell with a new protagonist named Mel. Cave Johnson was to replace GLaDOS as the antagonist, and Mel was going to have many different personality spheres follow her (such as the Adventure Sphere, Betty, and one referred to as the "Morgan Freeman Sphere") instead of just Wheatley. Play testers were up for the idea of Mel until they became disappointed when GLaDOS woke up and didn't recognize the player as the character that defeated her in the first game. Mel was then put in the multiplayer before being scrapped entirely and replaced with Atlas and P-Body.

According to Portal 2 writer Erik Wolpaw, Valve originally planned to include a gel that let you walk on walls.

Unfortunately, players would become queasy with its use. Valve tried reducing the frame rate and camera movement, but were eventually forced to drop the feature entirely.

Pneumatic Diversity Vent was introduced in a trailer at E3 as a way to move stuff around like turrets. However nowhere in the game can it be found. It was cut because the puzzle aspect was very limiting.

In test chamber 8, GLaDOS will leave shortly after you start the test to fix the turbines. Before leaving, she will give you sped up instructions. If you slow down what she says, you will hear she is actually quoting a line from Moby Dick.

In the final Doug Rattmann den in Portal 2, which is accessible through chapter 3 in test 17, if you go near the graffiti art you can hear somebody rambling through the wall, which is believed to be Doug himself.

When the Companion Cube is destroyed, you can hear human screaming as it disintegrates. This, and GLaDOS' insistence that the cubes do not talk in the first game, as well as Doug Rattman being able to hear the cube speak, suggest that the cubes contain people.

In Chamber 7, there is some sort of robotic singing coming from the Companion Cubes.

The Turret Opera, the song that was played at the end of the game, was sung entirely by Ellen McLain, who voiced both GlaDOS and the turrets. When recording the song, Ellen was asked to make up some words to put in the song, which she did using her "very bad high school Italian."

There is an achievement/trophy in the game called Ship Overboard. In the back of an office in Chapter 6: The Fall, Spoiler:there is a secret door hiding a hallway which leads to a dry dock containing a life preserver from The Borealis, a scientific research vessel owned by Aperture Science. The Borealis was first featured in Half Life 2 Episode 2. Getting close to and looking at the life preserver will get you the achievement.

The paintings in Chell's room at the beginning of the game changes from a day scene to a night scene depending on whether it is daytime or nighttime.