The histone acetyltransferase TIP60 is a coregulator of transcription factors and is implicated in tumorigenesis. In this study, we explored potential regulatory relationships between TIP60 and the c-Myb oncoprotein in hematopoietic cells. We first showed that TIP60 is a c-Myb interacting protein and that the interaction is dependent on the TIP60 acetyltransferase domain and c-Myb transactivation domain. We then found that coexpressing TIP60 decreases the transcriptional activation ability of c-Myb in functional reporter assays. A ChIP assay also revealed that TIP60 binds to the c-Myb target gene c-Myc promoter in a c-Myb-dependent manner. Consistently, knockdown of Tip60 expression by siRNA increased endogenous c-Myc expression. Furthermore, coimmunoprecipitation of Jurkat cell lysates revealed that c-Myb is associated with histone deacetylases HDAC1 and HDAC2, known to interact with TIP60 and repress transcription. Finally, we compared Tip60 expression in six primary AML samples with three normal CD34(+) cell samples using quantitative RT-PCR. Tip60 expression was significantly (∼60%) lower in the AML samples. In summary, these studies demonstrate that TIP60 negatively modulates c-Myb transcriptional activity by recruiting histone deacetylases in human hematopoietic cells, leading us to hypothesize that TIP60 is a normal regulator of c-Myb function and that dysregulated or mutated TIP60 may contribute to c-Myb-driven leukemogenesis.