Two pupils were treated for "serious injuries" at Alder Hey children's hospital, in Liverpool, and eight others had been taken to surrounding hospitals after their bus overturned on the way to school in Merseyside.
Emergency services declared a "major incident" after the morning rush-hour incident on the M53.
The bus was on its way to Calday Grange and West Kirby grammar schools in Wirral when it overturned on the hard shoulder.
Investigators were trying to establish what caused the bus to flip on its side as police said the crash was not believed to have involved another vehicle, despite early reports.
Graeme Robson of Merseyside police said: "We understand there were 54 people, including the driver, on the bus and sadly we can confirm that the driver and a 14-year-old schoolgirl have died.
"Two children were taken from the scene to Alder Hey children's hospital. Fifty other children were taken to an emergency training centre where they were medically assessed and eight of those children have been taken to the Countess of Chester and Arrowe Park hospitals.
"Our thoughts and condolences are very much with their families and friends at this tragic time."
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