Kit receptor and its ligand stem cell factor (SCF) are critical regulators of mast cell production, proliferation, degranulation, and chemotaxis. In this study, we investigated how Fyn kinase regulates chemotaxis of mast cells toward SCF. On beta1-integrin engagement, Fyn-deficient (fyn(-/-)) mast cells displayed a striking defect in cell spreading and lamellipodia formation compared to wild-type mast cells. The hematopoietic-specific Src family kinases (Lyn/Fgr/Hck) were not required for initial SCF-induced cell spreading. Reduced SCF-induced activation of Rac1 and Rac2 GTPases, p38 mitogen-activated protein kinase, and filamentous actin polymerization was observed in fyn(-/-) mast cells compared to wild-type mast cells. Retroviral-mediated expression of Fyn, constitutively active forms of Rac2 or phosphatidylinositol 3-kinase (PI3K) in fyn(-/-) mast cells rescued defects in SCF-induced cell polarization and chemotaxis of Fyn-deficient mast cells. Thus, we conclude that Fyn kinase plays a unique role upstream of PI3K and Rac GTPases to promote the reorganization of the cytoskeleton during mast cell spreading and chemotaxis.