Megakaryocytes express 2 NMII isoforms, NMIIA and NMIIB. At steady state, low RhoA/ROCK activity results in the localization of NMIIB, but not NMIIA, at the megakaryocyte cleavage furrow, a step required for the initiation of endomitosis. Subsequent downregulation of NMIIB expression by RUNX1 facilitates progression of endomitosis and polyploidization. Increasing RhoA activity and, subsequently, decreasing actin turnover in megakaryocytes results in the localization of both NMIIB and NMIIA at the cleavage furrow. Conversely, further inhibition of basal low RhoA activity increases actin turnover and leads to the loss of NMIIB from the cleavage furrow.