Macrophage colony-stimulating factor (M-CSF) has been found to be involved in multiple developmental processes, especially production of cells belonging to the mononuclear phagocyte system. The decision of myeloid progenitor cells to commit to differentiation depends on activation levels of the mitogen-activated protein kinases (MAPK), ERK1 and ERK2. Using the murine myeloid progenitor cell line FD-Fms, we show here that persistent activity of Src-family kinases (SFK) is necessary for FD-Fms cell differentiation to macrophages in response to M-CSF. Chemical inhibition of SFK blocked FD-Fms cell differentiation while it caused strong inhibition of the late phosphorylation of phospholipase C (PLC)-γ2 and MAPK. The PLC inhibitor U73122, previously shown to block M-CSF-induced differentiation, strongly decreased long-term MAPK phosphorylation. Interestingly, inhibiting SFK with SU6656 or the MAPK kinases MEK with U0126 significantly impaired development of mononuclear phagocytes in cultures of mouse bone marrow cells stimulated with M-CSF. Collectively, results support a model in which SFK are required for sustained PLC activity and MAPK activation above threshold required for commitment of myeloid progenitors to macrophage differentiation.