The Ets transcription factor PU.1, encoded by the gene Sfpi1, functions in a concentration-dependent manner to promote myeloid and B-cell development and has been implicated in myeloid and lymphoid leukemias. To determine the conse quences of reducing PU.1 concentration during hematopoiesis, we analyzed mice with two distinct hypomorphic alleles of Sfpi1 that produce PU.1 at ∼20% (BN) or ∼2% (Blac) of wild-type levels. Myeloid development was impaired in these mice, but less severely than in Sfpi1 null mice. To identify the down-stream target genes that respond to changes in PU.1 concentration, we analyzed ex vivo interleukin-3 dependent myeloid cell lines established from Sfpi1BN/BN, Sfpi1Blac/Blac and Sfpi1−/−fetal liver cells. Unexpectedly, many T-cell and natural killer cell genes were expressed in Sfpi1−/− cells and repressed in a dose-dependent manner in Sfpi1Blac/Blac and Sfpi1BN/BN cells. This pattern of dose-dependent T/NK-cell gene repression also occurred in ex vivo interleukin-7 dependent progenitor B cell lines. These results suggest that PU.1 functions in a concentration-dependent manner to repress T-cell and natural killer cell fates while promoting myeloid and B-cell fates.