The game opens with Layton explaining the witch trials of the medieval ages and how there weren't really any witches. Enter the Damsel in Distress, and then the pigeon that transforms into a clawed one-eyed thing that blows up the office and kidnaps their client.
Luke: What was that?! Layton: (calmly and with utter confidence): That, my dear boy, was a witch.
And a little later:
Luke: Espella's been kidnapped! Layton: [Flatly] Yes, that was careless of us.
Layton's Dull Surprise and ever-placid tone make for quite a few moments. After the culprit of the second case tells the defence they lost in the smuggest tone imaginable, the camera shows the defence bench. Layton's eyes are slightly narrowed and he looks thoroughly unimpressed and slightly peeved, while Phoenix is in the background, glaring intensely.
Wright greeting Layton: Not some normal conversation, but instead, WELCOME!, even with according, Objection-like speech bubble and a desk slam. And a cloud of flour, as he's working in the bakery.
When he's giving Layton some walnut bread, he slams the desk again (kicking up another flour cloud) and shouts TAKE THAT! at him, then points as the Objection theme plays. Maya tells him to cut it out, and he switches to embarrassed mode as the music dies.
Phoenix: I don't know why, but... I just get the urge to point and shout like that sometimes...
Luke and Layton's perplexed reactions to this.
The context also matters. At that point in the game you're just supposed to take refuge in some bakery with Espella, as you're hunted by the guards, and then the aforementioned WELCOME! bubble speech suddenly pops out, and you realize that Wright and Fey were the bakers.
Shortly after all this, Layton and Luke try and phrase "they're weird" in the most delicate way possible without actually saying "Fey and Wright are really friggin' weird".
Maya telling Phoenix "bad, Nick, bad!" like he's a badly behaved puppy she's getting after with a squirt bottle.
In the Special Episodes: Barnham working in the bakery. And even he fears Patty's wrath.
The start of the first Witch Trial, where Phoenix is told to introduce himself... "My name is Phoenix Wright, ace baker!" What's better? The line is fully voiced.
Phoenix: I could point at witnesses till the cows come home.
It gets lampshaded mercilessly throughout the game, culminating in a Tear Jerker/Crowning Moment Of Awesome/Crowning Moment of Funny scene where Layton apparently uses it to save Eve and Espella from falling to their deaths. It's only funny because how how much lampshading the entire pointing thing gets given over the game and how it gets used even more in this game than in either of the home series games.
Not to mention that the Judge continues to refer to the Vigilantes as skilled and professional despite the fact that two of them are blindly in love with a third, one of them can barely stand, one of them thinks he's testifying about the first witch trial and one of them locked the entire group inside of the bell tower by swallowing the key. True professionals, indeed.
Oddly enough, pressing one of the statements reveals that the Shakey, the aforementioned constantly-collapsing injured man is actually the fastest runner out of all of them, and was the first one to reach the scene and apprehend Espella. Phoenix is appropriately suprised.
At the end of this cross-examination, Phoenix finally breaks it and proves his point... leading to the ENTIRE LINE of witnesses being knocked over one at a time... all from a single finger point.
The end of the first part of the first witch trial. The case seems all but won, and then... There's a "HOLD IT!" in a voice you've never head before. It turns out to belong to... some guy in the gallery. No really, his actual name is listed as "Some Guy". He loudly and hammily proclaims himself to be the "fifth witness" who will singlehandedly-save this trial, and calls the other witnesses into a huddle. The Judge, Prosecution and Defence are all equally baffled.
Said "Some Guy" manages to be the cause of many, manyFunny Moments during the second half of the trial. Such as when he claims he heard a woman's voice call his name, which prompts the two female witnesses beside him to both loudly proclaim no woman would ever be interested in someone like him, to his face.
At the end of Chapter 3, there's a cutscene as a group of characters rush to the scene of a crime. Two of them are people you saw in front of the house a few moments ago, so that makes sense... but behind them is Emeer. There was no indication he was even in the area beforehand, he hasn't appeared at all since the last trial, and he's just suddenly there in the cutscene!
At the end of the first witch trial, Layton and Wright dramatically point together as Back-to-Back Badasses. For the second trial Layton is a golden statue, so it doesn't seem like this'll happen again... until the moment comes for the dramatic accusation, and Luke and Espella turn the statue in time with Phoenix's movements.
When the poor victim is brought in, the Judge has him added to evidence. Phoenix's commentary and expression sells it:
Phoenix: ( The poor professor who, while searching for evidence, became evidence...)
At one point in the third trial, Emeer interjects on one of Birdly the Bard's statements. If you choose to question him on this... it turns out he just wants to hire Birdly as his personal bard to sing the praises of the great Emeer Punchenburg the First. Birdly obliges. One overly long song later, and everyone in court relises that was pointless, and the Judge agrees to pretend it never happened.
It's even better if you didn't go down that path... because when Emeer returns to the witness stand later in the third trial, Birdly is standing at his side, constantly singing whenever he says something. And periodically insulting him as well. It's a Big Lipped Alligator Moment if you didn't see Emeer hire Birdly, which just makes it even funnier.
It's a little debatable if Cracker is ACTUALLY supposed to be saying them, since the actual sound effects are behind his speech and he's said to imitate the sounds perfectly; having him "say" them is just to put something in testimony text box. Still, it is rather amusing that they decided to have the parrot's voice actor say the sounds anyway.
Inquisitor Barnham valiantly attempts to stop Phoenix from inquiring about the Alchemist's neighbour, who turns out to be Emeer. The reaction when Emeer takes the stand, from both Barnham and Nick, is absolutely hilarious.
Phoenix: (Oh dearie ME.)
Similarly when you disprove his testimony:
Phoenix: That gem was useless. Barnham: Pssh. Just like everything else this witness says.
Pretty much every instance of Phoenix and Barnham tag-team insulting this character is comedy gold.
And then Maya notices something. In total, everyone at the reunion drank 17 cups of tea. So she thinks, maybe Layton has a rule about tea. Maybe he always has 17 cups of tea with every puzzle. Ace Attorney fans are doubled over in laughter about now. And then Phoenix quips that that particular Ace Attorney reference probably isn't appropriate, as the reference is about a big coffee drinker.
Phoenix has a new, cocky pose where he places his left hand on his hip and looks pleased with himself. It just oozes sass.
The cameo scene with Edgeworth at the end of the game. Also doubles as a moment of awesome.
It's dark humour...but it's still funny: When investigating The Legendary Fire, the team move to a vantage point to help them visualise how it must have appeared to one particular witness. Maya notes that to really put themselves in the witness' shoes, the whole town should be on fire. She then offers to go and get some matches.The Professor's reaction◊ is what makes it.
Eve's dramatics, and Mr. Cantabella's hospital chapter in the third special episode.
Constantine, Barnham's dog, delivers a few of these, especially with his vehement denials (via Luke's translations) that he is friends with Eve, Espella's cat. Gets doubly funny when, in the special episodes, when Barnham gives an almost identical denial over his obvious affection for Eve Belduke, a.k.a. Lady Darklaw. All present are deeply amused by the similarity.
When Lettie Mailer introduces herself as a courier, Phoenix Wright said, in his mind "A courier?" Espella then explained to him what a courier is before he told her he already knew that. After that, he said, in his own mind, "Stop misreading my mind."
A meta funny, but the professor's official height on the wiki is listed as 5'9", which is impossible because his shoulder is several inches lower than Phoenix's, meaning he should probably be closer to the heights of Apollo and Franziska (who are only a few centimetres apart in height, so the prof should be probably more like 5'4"). How is the professor 5'9"? They included his hat.
The entire scene where Layton is trying to explain puzzles to Maya and Phoenix, who are completely bemused by the concept most likely because they're still hypnotised at that point. As one youtuber puts it: "Phoenix can turn a case upside-down, Maya can use Spirit Channeling, and neither can solve a puzzle". Worth noting that neither of them have really even heard of what a puzzle even is up until now. Layton's obviously eager to introduce his new friends to the concept of puzzles, it's one of the rare times where his tone clearly changes from placid and calm to outright excitement, even if it's just text and beep-de-beeps. It's really cute.
Maya and Luke interacting like brother and sister as they do is both adorable as hell and funny; at one point, they come across glowing moss. Maya, predictably, pokes it and discovers it makes her finger glow. The next logical step for her is, obviously, cover Luke in moss for the sake of a Visual Pun, so he can be as "bright" as the professor. Luke reacts as you'd expect, and the amused Layton and Wright just leave them to their own devices. No points for guessing what happened in the end.
At one point, Maya has to wear a helmet (specifically, she filched Barnham's) to conceal her face. Phoenix tries valiantly to handwave it to the best of his ability on the spot, leading to this:
Phoenix: And um... don't comment on the helmet, please... She really wouldn't, err... like to have her face seen. How can I put this gently... It's not something you'd want to look at.
"The inquisition has ingeniously solved the query by disregarding it."
After beating the game, the bonus episodes have absoultely No Fourth Wall whatsoever.
Maya: Ease up, Nick! Sometimes a little self-awareness is fun. Isn't that right, avid video game player?
During the third trial when inquiring to Emeer about Layton's broken off golden arm, Phoenix can ask how much did he sell it for. There's silence in the court until a shocked Emeer asks if Phoenix plans to sell the whole body! Cue reactions of horror and derision from everyone around Phoenix.