Differentiation of hematopoietic cells from multipotential progenitors is regulated by multiple growth factors and cytokines. A prominent feature of these soluble factors is promotion of cell survival, in part mediated by expression of either of the anti-apoptotic proteins, BCL-2 and BCL-XL. The complex expression pattern of these frequently redundant survival factors during hematopoiesis may indicate a role in lineage determination. To investigate the latter possibility, we analyzed factor-dependent cell-Patersen (FDCP)-Mix multipotent progenitor cells in which we stably expressed BCL-2 or BCL-XL. Each factor maintained complete survival of interleukin-3 (IL-3)-deprived FDCP-Mix cells but, unexpectedly, directed FDCP-Mix cells along restricted and divergent differentiation pathways. Thus, IL-3-deprived FDCP-Mix BCL-2 cells differentiated exclusively to granulocytes and monocytes/macrophages, whereas FDCP-Mix BCL-XL cells became erythroid. FDCP-Mix BCL-2 cells grown in IL-3 were distinguished from FDCP-Mix and FDCP-Mix BCL-XL cells by a striking reduction in cellular levels of Raf-1 protein. Replacement of the BCL-2 BH4 domain with the related BCL-XL BH4 sequence resulted in a switch of FDCP-Mix BCL-2 cells to erythroid fate accompanied by persistence of Raf-1 protein expression. Moreover, enforced expression of Raf-1 redirected FDCP-Mix BCL-2 cells to an erythroid fate, and prohibited generation of myeloid cells. These results identify novel roles for BCL-2 and BCL-XL in cell fate decisions beyond cell survival. These effects are associated with differential regulation of Raf-1 expression, perhaps involving the previously identified interaction between BCL-2-BH4 and the catalytic domain of Raf-1.