Stefani shot a 6-under 65 at the Copperhead Course of the Innisbrook Resort and Golf Club to seize a two-shot advantage after 18 holes.
He earned his way onto the tour by finishing sixth on the money list of the Web.com Tour last year. Stefani had two victories on that circuit in 2012 and finally has a shot on the big stage at the age of 31.
Stefani played a bogey-free round on a course that is one of the most difficult tests on the tour.
Brian Harman bogeyed the final hole to shoot a 67 and was alone in second while Harris English, Tag Ridings, Peter Tomasulo and Brendan Steele were all at 68. English was tied for the lead heading to his last hole, but suffered a double bogey.
Only 25 members of the 156-man field broke par.