The cellular source of coagulation factor VIII (FVIII) remains controversial. Like many coagulation proteins, FVIII is produced in the liver, and FVIII synthesis has long been associated with hepatocytes. But extrahepatic synthesis also occurs, and mounting evidence suggests that hepatocytes are not responsible for FVIII production. To determine the tissue that synthesizes FVIII, we developed a Cre/lox-dependent conditional knockout (KO) model in which exons 17 and 18 of the murine factor VIII gene (F8) are flanked by loxP sites, or floxed (F8^F). In cells expressing Cre-recombinase, the floxed sequence is deleted, resulting in F8^F→KO gene inactivation. When F8^F mice were crossed with various tissue-specific Cre strains, we found that hepatocyte-specific F8-KO mice are indistinguishable from controls, whereas efficient endothelial-KO models display a severe hemophilic phenotype with no detectable plasma FVIII activity. A hematopoietic Cre model was more equivocal, so experimental bone marrow transplantation was used to examine hematopoietic FVIII synthesis. FVIII^null mice that received bone marrow transplants from wild-type donors were still devoid of plasma FVIII activity after hematopoietic donor cell engraftment. Our results indicate that endothelial cells are the predominant, and possibly exclusive, source of plasma FVIII.