BACKGROUND: One of the important biological activities of thrombopoietin (TPO) is to prevent the apoptosis of megakaryocytes. As the antiapoptotic protein Bcl-xL, which has been proven to be indispensable for erythroid differentiation, is also abundantly expressed in megakaryocytes, it is assumed that Bcl-xL plays an important role in megakaryopoiesis. OBJECTIVES: We investigated the expression of Bcl-xL during megakaryopoiesis and the underlying regulatory mechanism. METHODS AND RESULTS: In stem cell-derived megakaryocytes, expression of Bcl-xL increased in the early and mid-stages of the differentiation. Both in vitro in cell culture and in vivo in an animal model, expression of Bcl-xL protein was maintained until the platelet-producing stage. TPO depletion caused significant decrease in Bcl-xL protein level without affecting its mRNA in both megakaryocytes and TPO-dependent megakaryocytic UT-7/TPO cells, suggesting that Bcl-xL protein level in TPO-dependent cells is post-translationally regulated. In agreement with this finding, we recognized the appearance of a 12-kD fragment of Bcl-xL upon TPO depletion. This cleavage of Bcl-xL was inhibited by a caspase-3-specific inhibitor. Furthermore, pretreatment of UT-7/TPO with a phosphatidylinositol 3-kinase (PI3 K) inhibitor resulted in the cleavage of Bcl-xL even in the presence of TPO. We thus hypothesized that PI3 K inhibits the activation of caspase-3 and consequent cleavage of Bcl-xL. To prove this, we prepared UT-7/TPO cells transfected with constitutively active Akt-1. When TPO was depleted, the transfectant was significantly less liable to caspase-3 activation and Bcl-xL cleavage. CONCLUSIONS: Bcl-xL protein is expressed throughout megakaryopoiesis until platelets are produced, and its expression level is at least in part post-translationally regulated through TPO-mediated Akt activation.