Alternate Character Interpretation: Is Clive a tortured soul who did what he did because he saw it as the only solution, or an entitled brat who wanted to see the world suffer the same cruel fate that he did just so he could feel better about it?
Bill Hawks is painted as a totally unsympathetic Jerk Ass, but is there another, better side of him that helped him gain the favor of the people but which we never got to see?
Inferred Holocaust: A giant mecha bursts through an underground cavern and high above London, then proceeds to shoot at will on the city. Thousands presumably die. Then the same mecha falls through the hole it created and explodes in mid-air, completely incinerating the fake London underneath it, but apparently having no further effect on the city above, despite it actually being closer.
Dimitri's flashback in which he discovers Claire's dead body. All you see is her arm, but it's still unsettling, especially when one remembers this is a game for children.
A mentally unbalanced Clive using a mobile fortress to devastate London. It's not made clear if the area was evacuated, and if it wasn't, he may have killed a large number of people, although it's not brought up in-game.
Subject 3's backstory. He was a cute little bunny, abandoned and orphaned, before he was captured and sold to scientists and subjected to a number of torturous experiments.
Bill Hawks. The guy is essentially responsible for most of the game's conflict, exemplifying the trope Sleazy Politician that most people usually hate, yet you spend much of the game trying to rescue the bastard. And yet Clive somehow lets him get away with it, too!Also, he's ugly. He's particularly a popular Acceptable Target amongst the Top Ten List community.
Then again, he may have been an intentional Hate Sink right from the start; it's hard to tell.
The ending. First, getting to know why Clive did all this - he lost his parents in a time machine accident. Then, the scene where Claire says goodbye to the professor. Note: Layton actually cries and takes off his hat during this scene. And finally... Luke leaving because his father got a new job, which is even more sad after you've played the fourth game and realize that Layton is also losing one of his best friends - Clark.
This exchange, when Luke and Layton are parting ways and Luke begins to cry
Layton: Now Luke, a gentleman does not make a scene in public.
Luke: I'm sorry, Professor. I'm not a gentleman yet! *hugs him*
Layton:*gently* There, there.
Dmitri's flashback to the day Claire died, after he arrived too late. God, he looks so broken during the entire scene.
What Happened to the Mouse?: Subject 3's fate. Whether he is adopted and learns to accept humans, or is taken back to another facility, or just hangs out on the wharf for the rest of his life is unknown.