Within hematopoiesis C/EBPα is expressed specifically in both the granulocytic and monocytic myeloid lineages. Mice lacking C/EBPα have reduced neutrophil and monocyte production, due in part to diminished expression of the G-CSF, M-CSF, and GM-CSF receptor mRNAs. C/EBPα-ER directs the granulocytic differentiation of 32Dcl3 cells, and induction of late markers of granulopoiesis by C/EBPα-ER in 32Dcl3 cells was dependent upon its ability to slow G1 to S progression. We have now assessed the effect of C/EBPα-ER on the myeloid maturation of murine marrow progenitors. Regulation of the transgene using estradiol avoids biasing cell populations during transduction. Mononuclear cells isolated from 5-FU-treated mice were transduced with pBabePuro-C/EBPαER and selected for 2 days with puromycin. Surviving cells were isolated using a density gradient, subjected to lineage-depletion, which removed 98% of Mac1+ and Gr1+ cells, and placed in liquid or methylcellulose culture with IL3/IL6/SCF +/− estradiol. By 48 hrs, 80% of the cells had acquired myeloid surface markers. Mac1+/Gr1+ cells were granulocytes and Mac1+/Gr1− cells were monocytes, as confirmed by Wright’s-Giemsa staining of cytospins. Estradiol increased the proportion of monocytes 1.7-fold and reduced the proportion of granulocytes 2.5-fold in 5 independent assessments. Real-time PCR analysis indicated a 5-fold increase in CD14 and CD68/macrosialin RNA, a 2-fold increase in M-CSF receptor and PU.1 RNA, and a 4-fold decrease in NE RNA by 48 hrs. In contrast, estradiol had little effect on the development of monocytes or granulocytes from vector, pBabePuro-transduced cells. Consistent with these findings, in an initial experiment we observed increased granulocytes when progenitors from floxxed C/EBPα mice (kindly provided by P. Johnson) were transduced with pBabeNeo-CRE, compare with pBabeNeo, subjected to lineage-depletion, and cultured in IL3/IL6/SCF. Loss of C/EBPα was demonstrated by PCR and Western blotting. During the first 48 hrs after transfer of transduced wild-type cells to estradiol, C/EBPα-ER slowed the proliferation of myeloid progenitors 2.6-fold, increased their G1/S ratio 2.8-fold, and reduced myeloid CFUs 5.1-fold. CFU-M, CFU-G, and CFU-GM were affected equally. Transduction with C/EBPαL12V-ER, harboring a defective leucine zipper, or with C/EBPαBR3-ER, mutant in 4 basic region residues, slowed proliferation in liquid culture 2-fold, reduced myeloid CFUs 1.5-fold, and had little effect on the G1/S ratio, perhaps indicating a role for DNA-binding in the regulation of the cell cycle by C/EBPα in normal myeloid progenitors (CFUs) and precursors. The L12V variant did not affect lineage output, whereas C/EBPαBR3 reduced monocytic maturation 1.4-fold and increased granulocyte formation 1.4-fold, in 4 experiments. C/EBPαp30, expressed in a subset of AML cases and lacking an N-terminal transactivation domain, increased the G1/S ratio and reduced myeloid CFUs 1.6-fold and had no effect on myeloid maturation. In summary, exogenous C/EBPα increases the monocytic and reduces the granulocytic maturation of transduced myeloid progenitors cultured in cytokines whose receptors are not regulated by C/EBPα. Cell cycle inhibition may play a role in this lineage bias, as the the L12V and p30 variants had both a reduced ability to slow G1 progression and to induce monocytes. Induction of PU.1 may also favor monocytic maturation. Finally, results with BR3 compared with L12V, neither of which bind DNA, suggest that alteration of a protein interaction with the non-DNA contact surface of the C/EBPα basic region favors granulocyte formation.