The primary cellular source of factor VIII (FVIII) biosynthesis is controversial, with contradictory evidence supporting an endothelial or hepatocyte origin. LMAN1 is a cargo receptor in the early secretory pathway that is responsible for the efficient secretion of factor V (FV) and FVIII to the plasma. Lman1 mutations result in combined deficiency of FV and FVIII, with levels of both factors reduced to ∼10% to 15% of normal in human patients. We generated Lman1 conditional knockout mice to characterize the FVIII secretion profiles of endothelial cells and hepatocytes. We demonstrate that endothelial cells are the primary biosynthetic source of murine FVIII and that hepatocytes make no significant contribution to the plasma FVIII pool. Utilizing RiboTag mice and polyribosome immunoprecipitation, we performed endothelial cell–specific messenger RNA isolation and quantitative polymerase chain reaction analyses to confirm that endothelial cells highly express F8 and to explore the heterogeneity of F8 expression in different vascular beds. We demonstrate that endothelial cells from multiple, but not all, tissues contribute to the plasma FVIII pool in the mouse.