Granulocyte colony-stimulating factor (G-CSF) and fetal liver tyrosine kinase-3 (Flt3) ligand (FL) act in synergy to induce expansion and mobilization of hematopoietic progenitor cells. Regulation of mitogen activated protein (MAP) kinase pathways and gene transcription, induced by these cytokines were examined using the OCI-AML5 cell line. For this purpose, FL and G-CSF were used either alone, or in combination as the co-addition of FL and G-CSF (FL+G-CSF), or a chimeric molecule, progenipoietin-1 (ProGP-1). Both G-CSF and FL induced phosphorylation of extracellular signal-regulated kinases (ERKs) while p38 mitogen activated protein (MAP) kinase was phosphorylated only in response to G-CSF but not FL. Studies using specific kinase inhibitors suggested that both ERK and p38 MAP kinase pathways were required for the optimal cell proliferation in response to both G-CSF and FL. The magnitude of activation of the ERK pathway and induction of genes involved in cell cycle progression by G-CSF and FL exhibited a strong correlation with the degree of cell proliferation. These data suggest that OCI-AML5 cells proliferate at least in part, due to the activation of both ERK and p38 MAP kinase pathways in response to G-CSF and FL. This study represents the first report of the specific cell cycle genes induced by FL.