In human mast cells, derived from CD34(+) peripheral blood cells, we observed that Kit ligand (KL) failed to induce degranulation but acted in synergy with antigen to markedly enhance degranulation, levels of cytokine gene transcripts, and production of cytokines. Further examination revealed that antigen and KL activated common and unique signaling pathways to account for these varied responses. KL, unlike antigen, failed to activate protein kinase C but activated phospholipase Cgamma and calcium mobilization and augmented these signals as well as degranulation when added together with antigen. Both KL and antigen induced signals that are associated with cytokine production, namely phosphorylation of the mitogen-activated protein kinases, phosphatidylinositol 3-kinase-dependent phosphorylation of protein kinase B (also known as Akt), and phosphorylation of nuclear factor kappaB (NFkappaB). However, only KL stimulated phosphorylation of signal transducer and activator of transcription 5 (STAT5) and STAT6, whereas antigen weakly stimulated the protein kinase C-dependent induction and phosphorylation of c-Jun and associated activating protein-1 (AP-1) components, an action that was markedly potentiated by costimulation with KL. Interestingly, most signals were down-regulated on continuous exposure to KL but were reactivated along with cytokine gene transcription on addition of antigen. The findings, in total, indicated that a combination of FcepsilonRI and Kit-mediated signals and transcriptional processes were required for optimal physiologic responses of human mast cells to antigen.