Hematopoietic cytokines and intracellular signals emanating from their cell surface receptors support the proliferation, viability and differentiation of bone marrow cells. The precise role of cytokine/cytokine receptor interactions in lineage-restricted hematopoietic differentiation is incompletely defined. To study lineage specificity of cytokine signal transduction in terminal hematopoietic differentiation, we examined EpoR function during granulocytic differentiation. A cell culture model for granulocyte-macrophage colony stimulating factor (GM-CSF)-mediated granulocytic differentiation was used to determine the ability of Epo to support terminal differentiation of committed myeloid cells, engineered to ectopically express the full-length EpoR by retrovirus-mediated gene transfer. The EpoR was introduced into a multipotential murine cell line EML, from which GM-CSF-responsive, promyelocytic EPRO cells were generated in vitro. Ectopic expression of EpoR in transduced EML cells and myeloid EPRO cells was confirmed by immunoprecipitation of EpoR and Western blotting. We investigated the function of ectopic EpoR expression in myeloid EPRO cells to determine whether Epo/EpoR interaction can mediate granulocytic differentiation and the induction of myeloid gene expression. In EPRO cells expressing EpoR, Epo supported morphologic differentiation into neutrophils in a manner similar to GM-CSF. These results indicate that Epo can deliver differentiative signals along a nonerythroid lineage, functionally replacing GM-CSF receptor signals that induce granulocytic differentiation in this system. Morphologic granulocytic differentiation in response to Epo was associated with induction of surface CDllb/CD18 (Mac-1) expression, a myeloid marker detected by flow cytometry. To determine whether EpoR signals can functionally mediate activation of myeloid gene expression in differentiating EPRO cells, we examined the ability of Epo to induce myeloid-specific gene expression. Northern analysis showed that Epo-induced granulocytic differentiation was associated with activation of expression of genes encoding neutrophil specific granule proteins lactoferrin and gelatinase as well as myeloid transcription factor C/EBP e. These findings provide evidence for interchangeability of cytokine receptor signals that mediate terminal differentiation of committed myeloid cells and myeloid gene expression.