In most myeloid leukemias induced in mice by γ-radiation, one copy of chromosome 2 has suffered a deletion. To search for a potential tumor suppressor gene in that region, we have delineated the deletions in a panel of these tumors. A commonly deleted region of 2 megabase pairs (Mbp) includes the gene encoding the PU.1 transcription factor, a powerful inducer of granulocytic/monocytic differentiation. Significantly, in 87% of these tumors the remaining PU.1 allele exhibited point mutations in the PU.1 DNA binding domain. Surprisingly, 86% of these mutations altered a single CpG, implicating deamination of deoxycytidine, a common mutational mechanism, as the origin of this lesion. The “hot spot” resides in the codon for a contact residue essential for DNA binding by PU.1. In keeping with a tumor suppressor role for PU.1, enforced expression of wild-type PU.1 in the promyelocytic leukemia cells inhibited their clonogenic growth, induced monocytic differentiation, and elicited apoptosis. The mutant PU.1 found in tumors retained only minimal growth suppressive function. The results suggest that PU.1 normally suppresses development of myeloid leukemia by promoting differentiation and that the combination of gene deletion and a point mutation that impairs its ability to bind DNA is particularly leukemogenic.