In hematopoiesis, self-renewal, proliferation, differentiation and apoptosis represent opposing decisions made by stem cells and progenitor cells, which when dysregulated can result in leukaemia. Here, we have investigated the function of Myb proteins in regulating these key cellular decisions, using the cell line FDCP-mix A4 as a model of early hematopoiesis. High concentrations of IL-3 in these cells favour self-renewal over differentiation and apoptosis. However when endogenous Myb activity was inhibited with an inducible dominant interfering protein, self-renewal was replaced by apoptosis and differentiation. Differentiation was to granulocytes and monocyte/macrophages and was closely associated with a G1-S phase block in the cell cycle. As for normal hematopoiesis, cytokine-induced terminal differentiation of FDCP-mix cells is associated with concomitant proliferation prior to its completion. However, when Myb activity was inhibited during this process, proliferation and survival were both reduced, resulting in a much lower yield of mature cells. These results indicate multiple cellular roles of Myb proteins during normal hematopoiesis.