The events in the Bobby & Rufus flashbacks take place during season 4 of Supernatural.
When Sam introduces himself and Dean as FBI agents, he calls them Agent Biehn and Agent Reiser. These are the surnames of the actors who played Hicks (Michael Biehn) and Burke (Paul Reiser) in Aliens (1986).
In the flashbacks, Bobby and Rufus' FBI names are Riggs and Murtaugh. These are the main characters in Lethal Weapon (1987) (played by Mel Gibson and Danny Glover).
In addition to Bobby and Rufus using the main characters names from Lethal Weapon as their FBI names, Sam also quotes the movie by saying hes getting too old for this, which is a famous quote from the movie.
This is the first appearance of Steven Williams as Rufus since S7 E10 Deaths Door. This is also the first appearance of Rufus as himself and not a hallucination since his death in S6 E16 And Then There Were None.
The hospital shown in this episode is named St. Eligius, possibly named after the 1982 TV drama series "St. Elsewhere", which took place in a St. Eligius Hospital.