PU.1 is a hematopoietic cell-specific ets family transcription factor. Gene disruption of PU.1 results in a cell autonomous defect in hematopoietic progenitor cells that manifests as abnormal myeloid and B-lymphoid development. Of the myeloid lineages, no mature macrophages develop, and the neutrophils that develop are aberrantly and incompletely matured. One of the documented abnormalities of PU. 1 null (deficient) hematopoietic cells is a failure to express receptors for granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage (GM)-CSF, and M-CSF. To elucidate the roles of the myeloid growth factor receptors in myeloid cell differentiation, and to distinguish their role from that of PU.1, we have restored expression of the G- and M-CSF receptors in PU.1-deficient cells using retroviral vectors. We have similarly expressed PU.1 in these cells. Whereas expression of growth factor receptors merely allows a PU.1-deficient cell line to survive and grow in the relevant growth factor, expression of PU.1 enables the development of F4/80(+), Mac-1(+)/CD11b(+) macrophages, expression of gp91(phox) and generation of superoxide, and expression of secondary granule genes for neutrophil collagenase and gelatinase. These studies reinforce the idea that availability of PU.1 is crucial for normal myeloid development and clarify some of the molecular events in developing neutrophils and macrophages that are critically dependent on PU.1.