M-CSF is a cytokine essential for both the proliferation and differentiation of monocytes/macrophages. In this study, we established a new M-CSF-mediated differentiation-inducing system, and examined how the level and duration of the activation of ERK preceded M-CSF-mediated differentiation. TF-1-fms human leukemia cells rapidly proliferated in response to M-CSF. However, in the presence of a phorbol ester, TPA, TF-1-fms cells definitely switched their responsiveness to M-CSF from proliferation to differentiation, as evidenced by a more drastic morphological change and the appearance of cells with a higher level of phagocytic activity. In TF-1-fms cells expressing HIV-1 Nef protein in a conditionally active-manner, both M-CSF-mediated proliferation and M-CSF/TPA-mediated differentiation were inhibited by the activation of Nef. The Nef-active cells showed perturbed patterns of ERK activation. Under the proliferation-inducing conditions (TPA-free), parental or Nef-inactive cells showed modest ERK activation following M-CSF stimulation, whereas Nef-active cells showed an earlier and transient ERK activation, despite a decrease in their proliferation rate. Under the differentiation-inducing conditions, parental or Nef-inactive cells showed increased and prolonged ERK activation following M-CSF stimulation, whereas Nef-active cells showed transient ERK activation. These results supported the idea that the increased and prolonged ERK activation led to M-CSF-mediated macrophage differentiation but not to proliferation.