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In Everybody Loves a Clown, Jo first met Sam and Dean Winchester. Thinking they were intruders, she held Dean at gunpoint while Ellen apprehended Sam; Dean disarmed Jo, but she punched him in the face and took her gun back. Despite this rocky start, they became friends when Ellen realized they were John's sons and the Winchester brothers paid irregular visits to the Roadhouse.
In Born Under a Bad Sign, the demon Meg, while possessing Sam, tracked Jo down to the Sandpiper, a bar that she worked at in Duluth, Minnesota. She did not initially realize Sam was possessed and was uncomfortable about his presence from their last encounter and because of the strange shift in his personality: he mocked her about at the fact that she seemed to like Dean and said that Jo like a little sister to Dean. Then Sam tried to seduce her. He overpowered her and knocked her out when she rejected him. Jo woke up tied to a post. Meg forced her to recount Bill's death but claimed that what Ellen told her was not the truth: what really happened, Meg taunted, was that Bill survived with horrific injuries and John shot him dead to end his suffering.
Dean traced Meg's location via his cell phone. However, he refused to kill his brother to protect Jo and instead revealed that Sam was possessed. When Meg fled, Dean untied a shocked Jo and went after him. Jo saved Dean's life by bandaging his wound after Meg shot him and left him to drown, having found him using his cell phone ringtone. Remembering what Meg had said, she asked Dean if demons occasionally tell the truth as well as lies but did not reveal what she'd been told. She wanted to help him pursue Meg, but he refused.
Meg arrived to capture the Winchesters flanked by a pack of hellhounds. Dean shot at one, causing the pack to attack them. Jo saved Dean's life from a hellhound when she saw him knocked to the ground, but it cost her hers; she was injured to the point that she could not walk and bleeding heavily.
In Defending Your Life, when Dean Winchester was put on trial for his guilt by Osiris, Jo's ghost was summoned as a witness. Although Osiris failed to make her testify against Dean, Dean was pronounced guilty and she was compelled against her will to try to kill him in a manner reflecting her death: she turned the gas on the stove on, broke the window to blow away the ring of salt he was protecting himself with, and took his lighter to ignite the gas and kill Dean in an explosion. Sam Winchester temporarily incapacitated Osiris, however, freeing Jo from his control and causing her to vanish, but not before she tenderly touched his face.