"The Duke of Cambridge has today started work as an employee of Bond Air Services," Kensington Palace, his London residence, said in a statement.
"He will undertake job-specific training before he begins piloting missions for East Anglian Air Ambulance during the summer," it added.
The duke's programme will include using a flight simulator and in-flight training, the palace said.
Kensington Palace had announced William's new job in August last year and said he would be based at Cambridge airport in eastern England, responding to emergencies ranging from road traffic accidents to heart attacks.
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The 32-year-old's wife Kate is due to give birth next month. The couple already have a son, Prince George, who turns two in July.
They are expected to split their time between Kensington Palace and Anmer Hall, a country house on Queen Elizabeth II's private estate in Norfolk, eastern England.
While the prince's job will take up most of his time, his rota will take into account royal duties in Britain and abroad.
He is thought to be the first royal directly in line to inherit the throne to sign a job contract with a civilian employer.