Mice homozygous for the disruption of the PU.1 (Spi-1) gene do not produce mature macrophages. In determining the role of PU.1 in macrophage differentiation, the present study investigated whether or not there was commitment to the monocytic lineage in the absence of PU.1. Early PU.1−/− myeloid colonies were generated from neonate liver under conditions that promote primarily macrophage and granulocyte/macrophage colonies. These PU.1−/− colonies were found to contain cells with monocytic characteristics as determined by nonspecific esterase stain and the use of monoclonal antibodies that recognize early monocyte precursors, including Moma-2, ER-MP12, ER-MP20, and ER-MP58. In addition, early myeloid cells could be grown from PU.1−/− fetal liver cultures in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF). Similar to the PU.1 null colonies, the GM-CSF–dependent cells also possessed early monocytic characteristics, including the ability to phagocytize latex beads. The ability of PU.1−/− progenitors to commit to the monocytic lineage was also verified in vivo by flow cytometry and cytochemical analysis of primary neonate liver cells. The combined data shows that PU.1 is absolutely required for macrophage development after commitment to this lineage.