Family-Unfriendly Aesop: As happy as the ending is, it's hard to look past the fact that it came about because several characters refused to move on with their lives after a terrible tragedy. To the point that Henry and Angela faked a loving marriage for eighteen years, and adamantly refused to become the mask. (In fact, one of the main reasons Layton suspected that Angela was being impersonated was that the impersonator didn't realize she loved Randall more than Henry.) Worse, it's implied that if they had moved on and chosen to love each other, Randall's accusation that Henry stole his life would have been correct. It becomes weaker still when you factor in that Randall had amnesia and didn't remember his prior life until Descole forced the issue. If not for evil scheming, he would have had no reason to return, and Henry's dedication would have been for naught.
Fridge Logic: Lampshaded in the "Spinning Maize 3", post-puzzle description, which says that if ladybugs can fly, the two ladybugs should be able to find each other by doing so.
Harsher in Hindsight: After playing The Azran Legacy, it becomes pretty obvious that the men that accosted young Layton's father and drug him all the way to the Norwell Wall in chapter 2 were members of Targent. Layton's mother was telling the truth and had a right to freak out, while his father was probably lying to make sure Layton didn't worry. Considering the implication that the Laytons moved around a lot, they probably knew at least SOMETHING was after their son and didn't want to keep him in danger.
A pretty unintentional one with Pokémon Black 2 and White 2. Monte d'Or was built over the Akbadain Ruins in the middle of a desert and has a place of high commerce called Chance Avenue that grew with time. Meanwhile, Join Avenue also grows with time, is located right next to a desert, and is pretty close to some ruins that might as well be buried right underneath it (Nimbasa City then could be similar to Monte d'Or). There's even a Backpacker named Randall in Relic Passage, for crying out loud.
Mood Dissonance: The Masked Gentleman having Luke hanging at a great height and daring Layton to save him... a little of the seriousness is taken down because Luke is hanging from his shorts with his underwear clearly visible.
Narm: Chapter 2 is a flashback, recounting events that Layton is telling Luke and Emmy. It ends with Randall revealing an incredibly long and difficult series of equations he'd been working on. The story immediately cuts back to Emmy narrating about how Layton's tale ended on a horrifying note. While this is because the full story has not yet been revealed to the player, it comes across as a Faux Horrific account of a particularly difficult puzzle.
Layton, after seeing Randall fall to his apparent death, opens the door and sees a treasure trove, not the many archaeological finds he'd hoped to get, and sadly declares that this is what he gave his life to get. Granted, it later turns out that there was one more mystery Layton and Randall hadn't uncovered, but Layton's despair and grief are heart-rending.
The ending of the "Snow Bunny" play- the bunny saves the boy he's friends with, at the cost of his own life.
That One Puzzle: #112, A Duck of Tiles, has proven to be this for many players. You have to overlay a series of differently-colored tiles in a given space to form the picture of a duck. This is extremely difficult (it's worth 70 picarats, after all), much more than appears to be at first glance. There are other tile-based puzzles, but they're not nearly as hard as this one.
That One Sidequest: Some of the Rabbit Theater puzzles can be quite frustrating, because it's often unclear what the difference between two actions is (Do you want to show shock or awe?) and a puzzle may require one you don't have with little indication to show this. They can also become tedious because you can't make the performances go by faster. This is particularly annoying when you're trying to figure out the correct endings to long plays.