The successful test is the first in a series of planned STOVL-mode flights that will include short takeoffs, hovers and vertical landings.
"The joint F-35 industry and government team has already shown during extended ground tests that the STOVL propulsion system performs well, and thousands of hours of component testing has validated its durability," Dan Crowley, Lockheed Martin executive vice president and F-35 program general manager, said.
"Now we are seeing early proof that the system operates in flight as our team predicted."
The aircraft is powered by a single Pratt & Whitney F135 engine driving a Rolls-Royce LiftFan.
The system, which includes a Rolls-Royce three-bearing swivel duct that vectors engine thrust and under-wing roll ducts that provide lateral stability, produces more than 41,000 pounds of vertical thrust.
The F135 is the most powerful engine ever flown in a fighter aircraft.
F-35 Lead STOVL Pilot Graham Tomlinson of BAE Systems took off on the test flight, climbed to 5,000 feet and engaged the shaft-driven LiftFan propulsion system at 210 knots (288 mph), then slowed to 180 knots (207 mph) with the system engaged before accelerating to 210 knots and converting back to conventional-flight mode.
The STOVL propulsion system was engaged for a total of 14 minutes during the flight.
STOVL-mode flights will continue, with the aircraft flying progressively slower, hovering, and ultimately landing vertically.
The F-35B will replace US Marine Corps AV-8B STOVL fighters, F/A-18 strike fighters and EA-6B electronic attack aircraft.
The UK's Royal Air Force and Royal Navy, as well as the Italian Air Force and Navy, also will employ the F-35B.