"We can say that after a long night, there's a new dawn now for the Western Balkans," Rehn told a news conference.
Croatia's negotiations were launched in the early hours of Tuesday, after UN war crimes prosecutor Carla del Ponte said that Croatia's cooperation with the Hague war crimes tribunal was full.
Rehn said the opening of the negotiations had "erased any doubts" regarding the closer EU integration of Serbia and Montenegro, Macedonia, Bosnia and Herzegovina, and Albania.
He added the "the negotiations will be strict and fair," acknowledging "enlargement fatigue" in western Europe.