Endomitosis is the process by which mammalian megakaryocytes become polyploid during terminal differentiation. As in other endoreplicating cells, cyclin-cdk complexes are distinctly regulated, probably to overcome the strict mechanisms that prevent rereplication in most somatic cells. We have asked whether key factors involved in the assembly and licensing of replication origins are equally regulated during endomitosis. Cdc6, cdt1, and geminin expression was analyzed during differentiation of two human megakaryoblastic cell lines, HEL and K562, which respectively do and do not establish endoreplication cycles. Geminin was downregulated, whereas cdt1 levels were maintained upon differentiation of both cell lines, independently of whether cells entered extra S-phases. In contrast, cdc6 was present and remained nuclear only in differentiated endoreplicating cells. Interestingly, cdc6 protein expression was reestablished in K562 cells that underwent endomitosis after transient or stable cyclin E overexpression. The high levels of cyclin E reached in these cells appeared to influence the stabilization of cdc6 protein rather than its RNA transcription rate. Finally, cdc6 overexpression drove HEL cells into endoreplication cycles in the absence of differentiation stimuli. Our results show that both cdt1 and cdc6 are differentially regulated during megakaryocytic differentiation and suggest an active role of cdc6 in endomitosis.