In the hematopoietic cell system, the oncoprotein Ski dramatically affects growth and differentiation programs, in some cases leading to malignant leukemia. However, little is known about the interaction partners or signaling pathways involved in the Ski-mediated block of differentiation in hematopoietic cells. Here we show that Ski interacts with PU. 1, a lineage-specific transcription factor essential for terminal myeloid differentiation, and thereby represses PU. 1-dependent transcriptional activation. Consistent with this, Ski inhibits the biological function of PU. 1 to promote myeloid cells to differentiate into macrophage colony-stimulating factor receptor (M-CSFR)-positive macrophages. Using a Ski mutant deficient in PU. 1 binding, we demonstrate that Ski–PU. 1 interaction is critical for Ski's ability to repress PU. 1-dependent transcription and block macrophage differentiation. Furthermore, we provide evidence that Ski-mediated repression of PU. 1 is due to Ski's ability to recruit histone deacetylase 3 to PU. 1 bound to DNA. Since inactivation of PU. 1 is closely related to the development of myeloid leukemia and Ski strongly inhibits PU. 1 function, we propose that aberrant Ski expression in certain types of myeloid cell lineages might contribute to leukemogenesis.