The molecular mechanisms by which binding of monocyte/macrophage colony-stimulating factor to its receptor c-Fms promotes replication in primary macrophages are incompletely understood, as all previous studies involved overexpression of receptor mutants in transformed cells not endogenously expressing the receptor. To address this issue we retrovirally expressed, in bone marrow-derived macrophages, a chimeric receptor containing a range of tyrosine to phenylalanine mutations in the c-Fms cytoplasmic tail. We measured incorporation of bromodeoxyuridine as a marker of proliferation and phosphorylation of ERKs, Akt, and the receptor itself. Our data indicate that tyrosine 559 is the major mediator of receptor activation and cell death, intracellular signaling, and cell proliferation and that the tyrosine residues at positions 697 and 807 play lesser roles in these events. Importantly, we find that activation of the ERK and Akt pathways is necessary but not sufficient for induction of macrophage proliferation. Using specific small molecule inhibitors we find that a combination of the Src family kinase, phosphatidylinositol 3-kinase/Akt, phospholipase C, and ERK pathways mediates macrophage proliferation in response to M-CSF.