Myb family proteins are ubiquitously expressed transcription factors. In mammalian cells, they play a critical role in regulating the G(1)/S cell cycle transition but their role in regulating other cell cycle checkpoints is incompletely defined. Herein, we report experiments which demonstrate that c-Myb upregulates cyclin B1 expression in normal and malignant human hematopoietic cells. As a result, it contributes directly to G(2)/M cell cycle progression. In cell lines and primary cells, cyclin B1 levels varied directly with c-Myb expression. Chromatin immunoprecipitation assays, mutation analysis, and luciferase reporter assays revealed that c-Myb bound the cyclin B1 promoter preferentially at a site just downstream of the transcriptional start site. The biological significance of c-Myb, versus B-Myb, binding the cyclin B1 promoter was demonstrated by the fact that expression of inducible dominant negative c-Myb in K562 cells accelerated their exit from M phase. In addition, expression of c-Myb in HCT116 cells rescued cyclin B1 expression after B-myb expression was silenced with small interfering RNA. These results suggest that c-Myb protein plays a previously unappreciated role in the G(2)/M cell cycle transition of normal and malignant human hematopoietic cells and expands the known repertoire of c-myb functions in regulating human hematopoiesis.