The c-Kit receptor protein-tyrosine kinase plays a critical role in the differentiation, growth and survival of mast cells. Binding of its ligand stem cell factor (SCF), induces c-Kit dimerization, autophosphorylation, and recruitment of signaling proteins. The juxtamembrane sequence of c-Kit contains recruitment sites for the Src family kinases Fyn and Lyn, as well as Shp1 and Shp2 protein-tyrosine phosphatases. To characterize the role of Fyn in c-Kit signaling, we generated bone marrow-derived mast cells (BMMCs) from wild-type and Fyn knock-out mice. In contrast with previous studies of Lyn-deficient BMMCs, SCF treatment of Fyn-deficient BMMCs revealed no overt defects in the overall pattern of tyrosine phosphorylation, phosphatidylinositol 3' kinase recruitment to c-Kit, or phosphorylation of Stat3 transcription factor. However, Fyn-deficient mast cells showed a significant reduction in phosphorylation of Shp2 phosphatase and p38 mitogen-activated protein kinase. Defects in Shp2 and p38 phosphorylation were restored in Fyn-deficient mast cells transduced with a Fyn-expressing retrovirus (Fyn-rescue). Fyn-deficient BMMCs displayed reduced chemotaxis towards SCF, and this defect was corrected in Fyn-rescue cells. This study provides evidence that recruitment of both Shp2 and Fyn to juxtamembrane sites in c-Kit results in Shp2 phosphorylation, downstream signaling to p38 mitogen-activated protein kinase, and enhanced chemotaxis of mast cells.