Activation of the erythropoietin receptor is essential for the survival, proliferation, and differentiation of erythroid progenitors. To understand the role of erythropoietin receptor (EpoR) activation in erythroid differentiation, we infected primary erythroid progenitors with high-titer retrovirus encoding the non-hematopoietic prolactin receptor. The infected progenitors responded to prolactin in the absence of Epo by generating fully differentiated erythroid colonies. Therefore, differentiation of erythroid progenitors does not require an intracellular signal generated uniquely by the EpoR; the EpoR does not have an instructive role in erythroid differentiation. We also infected primary erythroid progenitors with retrovirus encoding chimeric receptors containing the extracellular domain of PrlR and the intracellular domain of either the wild-type or truncated EpoRs. A chimeric receptor containing only the membrane-proximal 136 amino acids of the EpoR cytoplasmic domain efficiently supported prolactin-dependent differentiation of erythroid progenitors. Substitution of the single cytoplasmic domain tyrosine in this receptor with phenylalanine (Y343F) eliminated its ability to support differentiation. The minimal EpoR cytoplasmic domain required for erythroid differentiation is therefore the same as that previously reported to be sufficient to support cell proliferation (D'Andrea, A. D., Yoshimura, A., Youssoufian, H., Zon, L. I., Koo, J. W., and Lodish, H. F. (1991) Mol. Cell. Biol. 11, 1980-1987; Miura, O., D'Andrea, A. D., Kabat, D., and Ihle, J. N. (1991) Mol. Cell. Biol. 11, 4895-4902; He, T.-C., Jiang, N., Zhuang, H., Quelle, D. E., and Wojchowski, D. M. (1994) J. Biol. Chem. 269, 18291-18294).