PU.1 is a transcription factor shown to regulate the expression of many important genes in myeloid and B cells. At birth, mice homozygous for the disruption of the PU.1 gene have erythrocytes, megakaryocytes, and T cells, but no mature myeloid or B cells. Cells with an inability to develop to maturity were found in this mouse for B cells, neutrophils, eosinophils, mast cells, and monocytes. Rescue of early monocytic cells by transfection with the PU.1 gene results in renewed development to macrophages. These results demonstrate that PU.1 is an important regulator in the development of cells in the hematopoietic system. J. Leukoc. Biol. 66: 727–732; 1999.