After Dean confides in Lucifer, who he at that point believes to be Castiel, Lucifer touches his shoulder as Cas often does when attempting to comfort Dean. Dean looks freaked out and later talks to Sam about how something seems off about him. If you look at all the scenes in which Cas touches Dean's shoulder, he always touches his left one. Lucifer put his hand on his right one, subconsciously encouraging the idea in Dean's head that something is not right about his friend.
Shoshanna Stern (who plays Eileen Leahy) is deaf. Her first language was American Sign Language but she comfortably reads lips.
The photo of teenage Sam and Dean in Sam's memory box was created using actual childhood photos of Jared Padalecki and Jensen Ackles.
The sign that Sam uses as he tries to say "thank you", is actually the sign for "f*ck", or, in some deaf communities, "f*ck you".
Sam is revealed to know a little sign language, which he learned while in college. In real life both Jared Padalecki and Misha Collins have reportedly taught their children sign language.