During the game's first case "Turnabout Memories", defense attorney Marvin Grossberg makes several references to his hemorrhoids acting up, with one being that they are "doing the Harlem Shake". While the English localization of the game wrote in several pop culture references and memes that were popular at the time of its release in 2007, this reference to the Harlem Shake was not a reference to the 2013 viral dance since it did not exist yet, but was actually a reference to the pre-existing Harlem Shake dance that first originated in New York City in 1981. However, according to the official Ace Attorney series timeline, Trials and Tribulations takes place in 2019, and in the events of that game, the Turnabout Memories case takes place five years beforehand in 2014, which is coincidentally one year after the Harlem Shake meme went viral. This makes it plausible for unaware players to assume that Grossberg is referencing the viral dance in the time when it was relevant, unintentionally predicting the meme's popularity and ostensibly inflating the number of meme references in the English script.

Alternatively, the German translation of the game instead has Grossberg say his hemorrhoids are doing "den Ententanz", also known as the Chicken Dance, another fad dance originating from Switzerland in the 1950's.

Although it had never been stated that Mia Fey had won all of her completed trials, Shu Takumi considered this to be true when considering how the game's fourth episode "Turnabout Beginnings" (which canonically features the first trial for both Mia and Miles Edgeworth, the latter of whom had been stated to have a perfect win record in trials prior to the events of the first game) would be resolved:

The original Japanese version of Phoenix Wright: Ace Attorney - Trials and Tribulations features a scene where the character Spoiler:Pearl Fey mistakenly spills curry on a scroll. She does this because her mother gave her a direction in writing to “Put a splendid end to the master of the house”, but Pearl misreads the kanji “華麗” (pronounced Karei, meaning splendid or gorgeous) as “kare” which means curry. The English localization changes Spoiler:the note to say “Gravely roast the Master in the fires of Hades and bring our vengeance to fruition.” and has Pearl instead spill gravy on the scroll.

Tatsuo Iwanoto, the character designer of Phoenix Wright: Ace Attorney - Trials and Tribulations, has said Dahlia Hawthorne was one of the more challenging characters for him to design, because the instructions he was given by the developers contained a lot of contradicting character elements, specifically saying:

Hideki Kamiya, best known as the game director of Devil May Cry, voiced Godot in the Japanese version of Trials and Tribulations.

Upon first meeting Lisa Basil and hearing she's the company director, Phoenix will state "Sh-She's human?! She seems more like a ghost in a shell." This is a reference to the manga/anime Ghost in the Shell.

The HMDs that she and her employees wear seem to also be a reference to the scouters in Dragon Ball Z.

When Mia Fey thinks about Dahlia Hawthorne in the first case, she says "Well, we know whose milkshake brings all the boys to the yard." This is a reference to the notable lyrics in the Kelis song "Milkshake".

When the Judge gets scared by Furio Tigre's constant growling, he says "Ruh-roh!". This is a reference to Scooby-Doo's titular character who also says "Ruh-roh!".

During the second case when going to Ron DeLite's apartment, you'll find a fish bowl-like helmet and a tape recorder-like machine on one of the stands. This is a reference to the previous game Phoenix Wright: Ace Attorney: Justice For All where Wendy Oldbag will be in a costume dressed as an alien like astronaut suit where those two pieces of costume are placed on the stand. Spoiler:Oldbag herself makes a cameo during the game's end credits in this costume.

In the third case, when asking Lisa Basil for more information on some evidence or profiles, this dialogue can occur.

Lisa: Sorry, that data is SuPer-Admin Restricted Desktop Access password-protected.
Maya: SuPer-Admin Restricted Desktop Access password-protected!? What!? This is madness!
Phoenix: No, Maya, that is SPARDA. She won't tell us unless we say the right code word.

The conversation is a reference to the infamous "This! Is! Sparta!" line from 300.

In the chapter "Bridge to Turnabout", Prosecutor Godot says that Maya's mind is like a "café au lait" in which Phoenix's words are the milk and the Judge is the spoon. Then, the Judge says "I-I'm a spoon!? I'm no spoony bard, I'll have you know!".

This is a reference to Tellah's famous line from Final Fantasy IV, "You spoony bard!".