Fibrinogen binding to integrin αIIbβ3 mediates platelet aggregation and requires agonist-induced “inside-out” signals that increase αIIbβ3 affinity. Agonist regulation of αIIbβ3 also takes place in megakaryocytes, the bone marrow cells from which platelets are derived. To facilitate mechanistic studies of inside-out signaling, we describe here the generation of megakaryocytes in quantity from murine embryonic stem (ES) cells. Coculture of ES cells for 8–12 days with OP9 stromal cells in the presence of thrombopoietin, IL-6, and IL-11 resulted in the development of large, polyploid megakaryocytes that produced proplatelets. These cells expressed αIIbβ3 and platelet glycoprotein Ibα but were devoid of hematopoietic stem cell, erythrocyte, and leukocyte markers. Mature megakaryocytes, but not megakaryocyte progenitors, specifically bound fibrinogen by way of αIIbβ3 in response to platelet agonists. Retrovirus-mediated expression of the reporter gene, green fluorescent protein, in ES cell-derived megakaryocytes did not affect viability or αIIbβ3 function. On the other hand, retroviral expression of CalDAG-GEFI, a Rap1 exchange factor identified by megakaryocyte gene profiling as a candidate integrin regulator, enhanced agonist-induced activation of Rap1b and fibrinogen binding to αIIbβ3 (P < 0.01). These results establish that ES cells are a ready source of mature megakaryocytes for integrin studies and other biological applications, and they implicate CalDAG-GEFI in inside-out signaling to αIIbβ3.