The transcription factor C/EBPα is an important mediator of granulocyte differentiation and regulates the expression of multiple granulocyte-specific genes including the granulocyte–colony-stimulating factor (G-CSF) receptor, neutrophil elastase, and myeloperoxidase. Indeed C/EBPα knockout mice display a profound block in granulocyte differentiation. To study this block in granulocytic differentiation in more detail, retroviral vector-mediated transduction of a dominant-negative retinoic acid receptor was used to establish hematopoietic growth factor–dependent, lympho-myeloid progenitor cell lines from the fetal livers of both the C/EBPα knockout animals (C/EBPα(−/−)) and their heterozygous littermates (C/EBPα(+/−)). Surprisingly, the C/EBPα(−/−) cell lines displayed significant spontaneous granulocytic differentiation, and this differentiation was markedly enhanced when the cells were stimulated with granulocyte macrophage (GM)–CSF. This GM-CSF–mediated differentiation was associated with the up-regulation of G-CSF receptor mRNA, and the combination of GM-CSF and G-CSF generated more than 95% mature neutrophils in the C/EBPα(−/−) cultures. The addition of all-transretinoic acid also enhanced this granulocytic differentiation of the cultured C/EBPα(−/−) cells, indicating that the activated retinoic acid receptors can enhance granulocytic differentiation through a molecular pathway that is independent of C/EBPα. These studies clearly indicate that terminal granulocytic differentiation associated with the up-regulation of C/EBPα-responsive genes can occur in the absence of C/EBPα, and they indicate the existence of multiple independent molecular pathways potentially used by primitive hematopoietic precursors that can lead to the development of mature granulocytes.