Megakaryocyte (MK) maturation involves DNA replication without cell division, resulting in polyploid (8N, 16N, 32N and 64N) cells ≤65μm in size. It has long been accepted that large, high ploidy MKs produce platelets; the cells required for normal blood clotting and wound repair post-injury. We report MKs within individual ploidies can be further sub-fractionated using antigens such as CD41, into large cytoplasmic MKs (LCM, CD41bright) and small cytoplasmic MKs (SCM, CD41dim). We demonstrate prospectively isolated LCM from RFP mice released significantly more (19-fold) RFP+ platelets than SCM following transplant into C57Bl/6 mice. Anti-platelet serum depletes mice of circulating platelets and during platelet recovery we found both a significant increase in high ploidy MK as well as LCM. In addition, in a mouse model (Pf4-Cre-Srsf3 knockout) with normal total MK numbers, but only ~10% of LCM compared to wildtype mice, severe thrombocytopenia resulted, with ~10% the number of platelets. We hypothesize LCM are the mature MKs responsible for platelet production and have undertaken single cell RNAseq and TEM studies to further elucidate the differences between these MK. Importantly, we have identified LCM and SCM in normal human samples. Together with our murine findings that ploidy alone is not sufficient for accurately predicting the platelet generating potential of a MK, our data suggests attempts to increase platelet formation and release for therapeutic benefit must not only drive high ploidy MK development, but must specifically target the generation of LCM.