The opening animation, featuring Josh and Tyler dressed as Luigi and fighting each other with vacuums.
Which is subverted in the sixth episode.
At the start, they compare Luigi winning the mansion in a shady competition to clicking on those "You are the one millionth visitor!" internet ad scams, prompting Josh to say "Luigi IS...the one millionth visitor!"
Tyler's game freezes for a while due to his Gamecube glitching, and to make it fair, Josh deliberately wastes a few seconds in the Gallery area to even it up...only for Tyler to then accidentally go to the Gallery himself and get stuck there for several minutes.
Also, Josh mentions that he was interrupted while sitting down to record the first episode by his new Gamecube being delivered.
Tyler hypothetically wearing a pinkJoshJepson shirt to a job interview. And getting hired.
Tyler: (as the employer) What does your shirt say? (as himself) "It's Not Supposed ToGo At An Angle". (as the employer) What's not supposed to go at an angle? (as himself) Your dick.
In a similar vein, Josh finds a secret in the Butler's Room (a sapphire in a bucket) and mentions he found a secret. Tyler acts like he didn't know about it to make Josh go Oh Crap!, but Josh says he didn't say what secret it was. Then Tyler says "but I bet you didn't know about this secret..." ...as he finds the same sapphire in a bucket.
Speaking of the Butler's Room, there's Tyler noticing a particular feature that the room boasts.
Josh has trouble capturing a Boo, getting it down to 1 HP only for it to constantly fly into different rooms, with him unable to suck it up fully for a while. He slowly gets angrier and angrier as this happens.
Tyler shocks Josh by telling him that he got a gold rank on every boss ghost in Area 2 except Bogmire. Josh then smugly tells him that he still has more money...only for Tyler to shock him again by telling him that he has about 1 million more.
In episode 4, Tyler has them play a version of the "Shag, Marry or Kill" game (with the options instead being "Handjob, Marry or Kill"), and then the groups of three people they have to decide over are all fellow Let's Players.
Tyler jokes about him and Josh being mentally in synch a lot in episode 6, at a time when (unbeknownst to both of them) they actually are doing almost exactly the same things at the same time on the screen.
The end of episode 6. Josh, trying to close the episode, says that he's been JoshJepson. Tyler declares that he's been Lucahjin. Josh yells at him, ending the episode with "YOU'RE A FUCKING IDIOT!"
Josh struggling to catch a boo stuck halfway in a wall.
Early in episode 7, Tyler finds a lump on a table and dubs it the "Table boner". Later, he comes back to the same room in search of a Boo, and...
Tyler: Wait... where'd the table boner go? The table boner's gone! What? WHAT? N—uh? Where'd the table boner go? That's not even right!
Josh: You sound very distraught.
Tyler: That's not even right, Josh! Not even right!
Josh finds the 50th gold bar literally one second after Tyler saying how unlikely it would be for him to find all 50 of them. (Though a little later it turns out Tyler was wrong and there are more than 50, it's still a bizarre coincidence).
Josh: Alright, alright! Get. In. My. Belly! And by belly I mean vacuum! [Beat, captures King Boo] YOU KNOW, IN THE VACUUM!
And now we have the Loser's Challenge video, which features Tyler dressed up as Luigi at PAX Prime, trying to find Mario. He eventually finds some random guy dressed as Mario and hugs him, asking where he's been. The very confused man responds "... Around?"
Also, on two occasions, Tyler stops just calling for Mario and instead stands in the middle of a crowded hallway and screams Mario's name at the top of his lungs. The second time, you can hear someone in the background actually reply "He's busy!"
At one point, Tyler asks a random person if he's seen Mario. The person suggests for Tyler to go to the washrooms and go down, a reference to warp pipes from the games. Tyler instead actually goes to the washrooms, walks inside the women's washroom, and quietly calls "...Mario?" before leaving awkwardly when he sees someone inside.
All of this is made better by Lucahjin (on the camera) absolutely pissing herself laughing.
Throughout the series, Tyler's obsession with 'the anteater room'. In the final follow-up video he even dismisses all Josh's suggestions for the best or funniest moment of the series, because nothing can compare to the anteater room.