Stem cell factor (SCF) binds to c-Kit and is an important mediator of survival, growth, and function of hematopoietic progenitor cells and mast cells. Lyn and other Src family members are activated by SCF and associate with phosphorylated tyrosine residues in the c-Kit juxtamembrane region. However, studies using c-Kit mutants incapable of directly recruiting Src family members suggest this kinase family plays a minimal role in c-Kit stimulus-response coupling mechanisms. The objective of this study was to specifically target Lyn and subsequently address its role in SCF-mediated responses of primary hematopoietic progenitor cells and mast cells. To this end, a dominant-inhibitory Lyn mutant and Lyn-deficient mice were used. Transfection of normal murine mast cells with kinase-inactive Lyn impaired SCF-induced growth. Further, SCF-induced proliferation and chemotaxis of Lyn-deficient mast cells were less than for wild-type mast cells. SCF-induced growth of progenitor cells lacking Lyn was also reduced compared with that of wild-type progenitor cells. Impairment of SCF-mediated responses of Lyn-deficient mast cells and progenitor cells did not result from reductions in surface expression of c-Kit. These studies demonstrate that Lyn is required for normal SCF-mediated responses of primary progenitors and for a differentiated lineage.