In mammals, platelets are produced in the blood by cytoplasmic fragmentation of megakaryocytes (MKs). Platelet production is thus dependent on both the MK number and size. During differentiation, MKs switch from a division by mitosis to polyploidization by endomitosis to increase their size. The endomitotic process includes several successive rounds of DNA replication with an entry in mitosis with a failure in late cytokinesis and a defect in karyokinesis. This leads to a giant cell with a modal ploidy at 16N and one multilobulated nucleus. The entire genome is duplicated several times and all alleles remain functional producing a hypermetabolic cell. A defect in abscission explains the cytokinesis failure and is related to an altered accumulation of actomyosin at the cleavage furrow as a consequence of both a low local RhoA activity and silencing of the MYH10 gene. This mechanism is regulated by transcription factors that govern differentiation explaining the intricacies of both processes. However, the endomitotic cell cycle regulation is still incompletely understood, particularly mitosis entry, escape to the tetraploid checkpoint, and defect in karyokinesis. Polyploidization is regulated during ontogeny, the first embryonic MKs being 2N. The molecular mechanism of this embryo-fetal/adult transition is beginning to be understood. In physiological conditions, MK ploidy is increased by an enhanced platelet demand through the thrombopoietin/myeloproliferative leukemia axis. In numerous hematologic malignancies, MK ploidy decreases, but it is always associated with a defect in MK differentiation. It has been proposed that polyploidization induction could be a treatment for some malignant MK disorders.