The bZip transcription factor MafB is expressed specifically in the myeloid lineage of the hematopoietic system and is up-regulated successively during myeloid differentiation from multipotent progenitors to macrophages. Here we report that this induction reflects an essential role of MafB in early myeloid and monocytic differentiation. We observed that the expression of MafB in transformed chicken hematopoietic precursors dramatically increases the proportion of myeloid colony formation at the expense of multipotent progenitor-type colonies. In addition, the overexpression of MafB in transformed myeloblasts stimulates the rapid formation of macrophages, as judged by morphology, surface marker expression and functional criteria. MafB-induced macrophages exhibit typical levels of phagocytic activity and nitric oxide release after activation by lipopolysaccharide. By contrast, overexpression of the myeloid transcription factor PU.1 in these cells does not induce macrophage differentiation. Furthermore, a dominant-negative allele of MafB inhibits both myeloid colony formation and the differentiation of myeloblasts into macrophages. Taken together, our results indicate that MafB induction is a specific and essential determinant of the monocytic program in hematopoietic cells.