Mast cells (MCs) participate in allergy, inflammation, and defense against pathogens. They release multiple immune mediators via exocytosis, a process that requires SNARE proteins, including syntaxins (Stxs). The identity of the Stxs involved in MC exocytosis remains controversial. Here, we studied the roles of Stx3 and -4 in fully developed MCs from conditional knockout mice by electrophysiology and EM, and found that Stx3, and not Stx4, is crucial for MC exocytosis. The main defect seen in Stx3-deficient MCs was their inability to engage multigranular compound exocytosis, while leaving most single-vesicle fusion events intact.