Neutrophil granulocytes (Gs) represent highly abundant and short-lived leukocytes that are constantly regenerated from a small pool of myeloid committed progenitors. Nuclear receptor (NR) family members are ligand-activated transcription factors that play key roles in cellular proliferation and differentiation processes including myelopoiesis. Retinoid X receptor alpha (RXRalpha) represents the predominant NR types I and II homo- and heterodimerization partner in myeloid cells. Here we show that human myeloid progenitors express RXRalpha protein at sustained high levels during macrophage colony-stimulating factor (M-CSF)-induced monopoiesis. In sharp contrast, RXRalpha is down-regulated during G-CSF-dependent late-stage neutrophil differentiation from myeloid progenitors. Down-regulation of RXRalpha is critically required for neutrophil development since ectopic RXRalpha inhibited granulopoiesis by impairing proliferation and differentiation. Moreover, ectopic RXRalpha was sufficient to redirect G-CSF-dependent granulocyte differentiation to the monocyte lineage and to promote M-CSF-induced monopoiesis. Functional genetic interference with RXRalpha signaling in hematopoietic progenitor/stem cells using a dominant-negative RXRalpha promoted the generation of late-stage granulocytes in human cultures in vitro and in reconstituted mice in vivo. Therefore, our data suggest that RXRalpha down-regulation is a critical requirement for the generation of neutrophil granulocytes.