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Knowles, who said her dark denim shorts were concealed by her baggy T-shirt, said the supervisor placed the antenna of his walkie-talkie between her legs.
"He said, 'I don't want to see your panties or anything, but do you have any on?'" Knowles said.
"I didn't want to show him anything. He wanted me to basically show him my crotch. I was completely humiliated. It was vulgar. It was macho. It was rude," she said.
Knowles said she was escorted off the plane and taken to a John F. Kennedy Airport hangar, where she showed workers she was wearing shorts.
She said she returned to the plane but a worker then told her she had to exit because the pilot was refusing to fly with her aboard.
JetBlue declined to comment.