Embryonic stem cells (ESCs) could potentially compensate for the lack of blood platelets available for use in transfusions. Here, we describe a new method for generating mouse ESC-derived platelets (ESPs) that can contribute to hemostasis in vivo. Flow cytometric sorting of cells from embryoid bodies on day 6 demonstrated that c-Kit(+) integrin alpha IIb (alpha IIb)(+) cells, but not CD31(+) cells or vascular endothelial cadherin(+) cells, are capable of megakaryopoiesis and the release of platelet-like structures by day 12. alpha IIb beta 3-expressing ESPs exhibited ectodomain shedding of glycoprotein (GP)Ibalpha, GPV, and GPVI, but not alpha IIb beta 3 or GPIb beta. ESPs showed impaired alpha IIb beta 3 activation and integrin-mediated actin reorganization, critical events for normal platelet function. However, the administration of metalloproteinase inhibitors GM6001 or TAPI-1 during differentiation increased the expression of GPIb alpha, improving both thrombogenesis in vitro and posttransfusion recovery in vivo. Thus, the regulation of metalloproteinases in culture could be useful for obtaining high-quality, efficacious ESPs as an alternative platelet source for transfusions.