The c-myb proto-oncogene is a key regulator of hematopoietic cell proliferation and differentiation. MYB mRNA is expressed at high levels in, and is required for the proliferation of, most human myeloid and acute lymphoid leukemias. Recently, chromosomal translocation and genomic duplications of c-MYB have been identified in human T-cell acute leukemia. The present work focuses on the effects of mutations in different domains of the murine c-Myb protein on its transforming ability as defined by suppression of myelomonocytic differentiation and continued proliferation. Using both a novel myeloid cell line-based assay and a primary hematopoietic cell assay, we have shown that mutation of single residues in the transactivation domain important for CBP/p300 binding leads to complete loss of transforming ability. We also simultaneously mutated residues in the DNA-binding domain and the negative regulatory domain of the protein. These double mutants, but not the corresponding single mutants, show a complete loss of transforming activity. Surprisingly, these double mutants show severely impaired transactivation and are also defective for CBP/p300 binding. Our results imply that multiple Myb domains influence its interaction with CBP/p300, highlight the importance of this interaction for myeloid transformation, and suggest an approach for molecular targeting of Myb in leukemia.