Noor Salman, 30, formerly of Fort Pierce, Florida, was charged by indictment with aiding and abetting Omar Mateen's attempted provision and provision of material support to the Islamic State of Iraq and the Levant (ISIL), a designated foreign terrorist organization, and obstruction of justice.
A federal judge in Oakland, Calif., ordered Noor Salman, 30, held pending a bail hearing February 1.
Salman was arrested on Monday in California, where she was living with her mother in the San Francisco area, according to her uncle, Al Salman, who denied she played a role in the attack.
A two-count indictment says that between April and June of past year, Salman "did knowingly aid and abet" her husband's attempt to support the Islamic State group. A detention hearing was set for two weeks from Wednesday. Federal prosecutors from Orlando want to try her in a Florida courtroom.
Investigators spent hours interviewing Salman after the shooting, trying to determine what she knew about.
It was Salman's second appearance in court since her arrest. The uncle said she was innocent and had no idea of her husband's plans.
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Mateen called a local TV station and 911 to "pledge allegiance" to the Islamic State during the siege at Pulse nightclub on June 12.
Her arrest came after she was interviewed numerous times by FBI agents investigating the June 12 attack in Florida.
Mateen killed 49 people and injured 53 others in what became the deadliest mass shooting in US history.
Salman's husband, Omar Mateen, died in a shootout with police after killing 49 club patrons.
Charles Swift, director of the Richardson, Texas-based Constitutional Law Center for Muslims in America, planned to represent Noor Salman at the Wednesday hearing, said public defender John Paul Reichmuth, who served as her attorney Tuesday. She will be transferred from Northern California to the Middle District of Florida, the Federal Bureau of Investigation said.
However her family and her attorney Linda Moreno insist the young woman - who grew up in California and who had a four-year-old son with Mateen - had no prior knowledge of the attack and was herself the victim of domestic abuse.