To assess cooperation between G-CSF signals and C/EBPα, we characterized Ba/F3 pro-B cell lines expressing C/EBPαWT-ER and the G-CSF receptor (GCSFR). In these lines, GCSFR signals can be evaluated independent of their effect on C/EBPα levels. G-CSF alone did not induce the MPO, NE, LF, or PU. 1 RNAs, and C/EBPαWT-ER alone stimulated low-level MPO and high-level PU. 1 expression. Simultaneous activation of the GCSFR and C/EBPαWT-ER markedly increased MPO and NE induction at 24 h, and LF mRNA was detected at 48 h. G-CSF did not increase endogenous GCSFR, endogenous C/EBPα or exogenous C/EBPαWT-ER levels, and C/EBPαWT-ER did not induce endogenous or exogenous GCSFR. Several GCSFR mutants were also co-expressed with C/EBPαWT-ER. Mutation of all four cytoplasmic tyrosines prevented NE induction but enhanced MPO induction. Mutation of Y704 was required for increased MPO induction. Consistent with this finding, removing IL-3 without G-CSF addition enabled MPO, but not NE, induction by C/EBPαWT-ER. GCSFR signals or related signals from other receptors may cooperate with C/EBPα to direct differentiation of normal myeloid stem cells.