The molecular mechanisms that regulate megakaryocyte (MK) ploidization are poorly understood. Using MK differentiation from primary human CD34⁺ cells, we observed that p19^INK4D expression was increased both at the mRNA and protein levels during ploidization. p19^INK4D knockdown led to a moderate increase (31.7% ± 5%) in the mean ploidy of MKs suggesting a role of p19^INK4D in the endomitotic arrest. This increase in ploidy was associated with a decrease in the more mature MK population (CD41^highCD42^high) at day 9 of culture, which was related to a delay in differentiation. Inversely, p19^INK4D overexpression in CD34⁺ cells resulted in a decrease in mean ploidy level associated with an increase in CD41 and CD42 expression in each ploidy class. Confirming these in vitro results, bone marrow MKs from p19^INK4D KO mice exhibited an increase in mean ploidy level from 18.7N (± 0.58N) to 52.7N (± 12.3N). Chromatin immunoprecipitation assays performed in human MKs revealed that AML-1 binds in vivo the p19^INK4D promoter. Moreover, AML-1 inhibition led to the p19^INK4D down-regulation in human MKs. These results may explain the molecular link at the transcriptional level between the arrest of endomitosis and the acceleration of MK differentiation.