The cell-cycle-regulated Myb-family transcription factor B-Myb is crucial during S phase in many diploid cell types. We have examined the expression and function of B-Myb in megakaryocytic differentiation, during which cells progress from a diploid to a polyploid state. In contrast to terminal differentiation of most haematopoietic cells, during which B-myb is rapidly downregulated, differentiation of megakaryocytes is accompanied by continued B-myb RNA and protein expression. Overexpression of B-Myb in a megakaryoblastic cell line resulted in an increase in the number of cells entering S phase and, upon induction of differentiation, the fraction of cells actively endoreplicating increased. By contrast, reduction of B-Myb levels using short interfering (si)RNA resulted in a decline in S-phase progression during both normal and endoreplicative DNA synthesis. This effect correlated with aberrant localisation of initiation of DNA replication within the nucleus and an increased fraction of cells in mitosis. Chromosomal fragmentation and other aberrations, including shorter, thicker chromatids, end-to-end fusion, and loss of a chromatid, suggest that reduced B-Myb activity is also associated with structural chromosomal instability.