Sam first meets Jo when he and Dean investigate a message on the dad's phone left by a women named Ellen. After they break into the Roadhouse, Ellen and Jo hold them at gunpoint until Ellen figures out their John's sons. They talk and find out Ellen and Jo know about the demon and knew John. At the end of the episode, Jo makes hints for Sam to go so that she can talk to his brother, Dean.
Soon after, Sam and Dean walk in on Jo and Ellen arguing about Jo wanting to be a hunter. Jo shows them the research shes done and Sam is impressed with it and wants to take the case, which they do. Sam later feels bad about stealing Jo's case. When Jo follows Sam and Dean on their case and lies to her mother, Sam isn't happy about it but treats her more like an equal and a professional than Dean does. After she goes missing, he is very concerned, but they get her back. Ellen finds out they lied to her and comes to collect them. Sam and Jo sit in the back of the Impala on the way back. Back at the Roadhouse, Ellen tells Jo that her dad was killed due to John's mistake. This upsets Jo and she stops talking to Sam and Dean.
Sam and Jo meet again when Sam becomes possessed by a demon and attacks her.
In "Good God, Y'all", Sam bumps into Jo when a town supposedly becomes overrun with demons. Thinking Sam is a demon, Jo captures Sam. Sam tries to convince Jo that he's' not a demon, but she doesn't believe him. Jo eventually releases Sam after Dean and Ellen convince her that Sam isn't a demon and there's something else going on there.
Sam and Jo are reunited in "Defending Your Life" when she is called to be a witness.
- Sam had been raised in the hunting lifestyle, but longed for normalcy and often fought with his father about it. In comparison, Jo led a fairly normal life but wanted to be a hunter against her mother's wishes. Each managed to get a taste of their dreams to tragic ends: Sam's girlfriend was killed by demons to push him back into hunting and Jo herself died on a mission to kill Lucifer.
- Both felt that they were seen as freaks.
- Both were former college students-turned-hunters.