This study was conducted to elucidate the anti-apoptotic mechanism operating during differentiation of megakaryocytes (Mgks) from hematopoietic stem cells. As previous reports have indicated the relevance of Bcl-xL for maintaining primitive hematopoietic cells, we focused on Bcl-xL among various anti-apoptotic proteins. Expression of Bcl-xL, which is relatively low in stem cells, increased gradually within 2–3 days of the culture of c-kit+/Lineage- stem cells in the presence of TPO. Expression of Bcl-xL temporally correlated well with that of GATA-1. Inhibition of GATA-1 expression in TPO-independent Meg-O1 megakaryocytic cells by using siRNA resulted in the decrease in Bcl-xL expression, suggesting that expression of Bcl-xL is directly or indirectly regulated by the GATA-1 transcription factor. On the other hand, when TPO was withdrawn from TPO-dependent UT-7 megakaryocytic cells, expression of Bcl-xL protein decreased dramatically within 48 hours despite the stable expression of Bcl-xL mRNA, indicating the post-transcriptional regulation of Bcl-xL after TPO depletion. As a broad caspase inhibitor z-VAD-fmk significantly reduced the decrease in Bcl-xL expression, we considered that proteolysis of Bcl-xL by a caspase might be responsible for down regulation of Bcl-xL. Indeed, corresponding with the time course of decrease in Bcl-xL protein after TPO depletion, activation of caspase-3 was recognized by immunostaining. As these results suggest that TPO-mediated signaling maintains caspase-3 inactive, we cultured UT-7 cells in the presence of a PI3K inhibitor or a MAPK inhibitor in search of the signaling molecule involved in this process. As PI3K inhibitor, wortmannin, induced the activation of caspase-3 even in the presence of TPO, we concluded that PI3K, in the downstream signaling pathway of c-Mpl, maintains caspase-3 inactive, which prevents cleavage of Bcl-xL by caspase-3. Withdrawal of TPO from primary culture of megakaryocytes similarly resulted in the activation of caspase-3 and decreased expression of Bcl-xL protein. Taken together, we conclude that expression of Bcl-xL protein is regulated transcriptionally by GATA-1 and post-transcriptionally by caspase-3 whose activation is prevented by TPO-mediated PI3K activation.