Apoptosis is a recognized limitation for generating large numbers of megakaryocytes in culture. The responsible apoptosis genes have been rigorously studied in vivo in mice, but are poorly characterized in human culture systems. As CD34+ cells isolated from human umbilical vein cord blood were differentiated into megakaryocytes in culture, two distinct cell populations were identified by flow cytometric forward and side scatter: larger size, lower granularity and smaller size, higher granularity. The larger size, lower granularity cells were CD41aHighCD42aHighphosphatidylserineLow, had an electron microscopic morphology similar to mature bone marrow megakaryocytes, developed proplatelets, and displayed a signaling response to platelet agonists. The smaller size, higher granularity cells were CD41aLowCD42aLowphosphatidylserineHigh, had a distinctly apoptotic morphology, were unable to develop proplatelets and showed no signaling response. Screening differentiating megakaryocytes for expression of 24 apoptosis genes identified BCL2L2 as a novel candidate megakaryocyte apoptosis regulator. Lentiviral BCL2L2 over-expression decreased megakaryocyte apoptosis, increased CD41a+ larger size, lower granularity cells and increased proplatelet formation by 58%. An association study in 154 healthy donors identified a significant positive correlation between platelet number and platelet BCL2L2 mRNA levels. This finding was consistent with the observed increase in platelet like particles derived from cultured megakaryocytes overexpressing BCL2L2. BCL2L2 also induced small, but significant increases in thrombin-induced platelet like particles αIIbβ3 activation and P-selectin expression. Thus, BCL2L2 restrains apoptosis in cultured megakaryocytes, promotes proplatelet formation and is associated with platelet number. BCL2L2 is a novel target for improving megakaryocyte and platelet yields in in vitro culture systems.