Mast cells play a critical role in host defense against a number of pathogens. Increased mast cell infiltration has been described in allergic asthma, in rheumatoid arthritis, and during helminthes infection. Despite the importance of mast cells in allergic disease and defense against infection, little is known about the mechanisms by which mast cells migrate to various tissues under steady state conditions or during infection or inflammation. Here, we show that activation of c-Kit by its ligand, stem cell factor (SCF), cooperates with α₄ integrin in inducing directed migration of mast cells on fibronectin. A reduction in migration and activation of a small G protein, Rac, was observed in mast cells derived from class I_A phosphoinositide-3 kinase (PI-3kinase)–deficient mice in response to SCF stimulation and in mast cells expressing the dominant-negative Rac (RacN17), as well as in mast cells deficient in the hematopoietic-specific small G protein, Rac2. In addition, a PI-3kinase inhibitor inhibited α₄- as well as SCF-induced migration in a dose-dependent fashion. In contrast, a mitogen-activated protein kinase (MAPK) inhibitor had little effect. Consistent with the pharmacologic results, abrogating the binding of the p85α subunit of class I_A PI-3kinase to c-Kit also resulted in inhibition of SCF-induced migration on fibronectin. These genetic and biochemical data demonstrate that both c-Kit and α₄ integrin signaling are linked to class I_A PI-3kinase and Rac pathways and regulate integrin-directed (haptotactic) migration in mast cells.