c-Kit and its ligand stem cell factor (SCF) are important for β-cell survival and maturation; meanwhile, interactions between the Fas receptor (Fas) and Fas ligand are capable of triggering β-cell apoptosis. Disruption of c-Kit signaling leads to severe loss of β-cell mass and function with upregulation of Fas expression in c-Kit(Wv/+) mouse islets, suggesting that there is a critical balance between c-Kit and Fas activation in β-cells. In the present study, we investigated the interrelationship between c-Kit and Fas activation that mediates β-cell survival and function. We generated double mutant, c-Kit(Wv/+);Fas(lpr/lpr) (Wv(-/-)), mice to study the physiological and functional role of Fas with respect to β-cell function in c-Kit(Wv/+) mice. Isolated islets from these mice and the INS-1 cell line were used. We observed that islets in c-Kit(Wv/+) mice showed a significant increase in β-cell apoptosis along with upregulated p53 and Fas expression. These results were verified in vitro in INS-1 cells treated with SCF or c-Kit siRNA combined with a p53 inhibitor and Fas siRNA. In vivo, Wv(-/-) mice displayed improved β-cell function, with significantly enhanced insulin secretion and increased β-cell mass and proliferation compared with Wv(+/+) mice. This improvement was associated with downregulation of the Fas-mediated caspase-dependent apoptotic pathway and upregulation of the cFlip/NF-κB pathway. These findings demonstrate that a balance between the c-Kit and Fas signaling pathways is critical in the regulation of β-cell survival and function.