Factor V (FV), a central regulatory protein in hemostasis, is distributed into distinct plasma and platelet compartments. Although platelet FV is highly concentrated within the platelet alpha-granule, previous analysis of human bone marrow and liver transplant recipients has demonstrated that platelet FV in these individuals originates entirely from the uptake of plasma FV. In order to examine further the biosynthetic origins of the platelet and plasma FV pools, we performed bone marrow transplantations of Fv-null (Fv-/-) fetal liver cells (FLCs) into wild-type mice. Fractionation of whole blood from control mice demonstrated that approximately 14% of total blood FV activity is platelet-associated. Mice that received transplants of Fv-null FLCs displayed a high degree of engraftment and appeared grossly normal, with no evidence for spontaneous hemorrhage. Although total FV levels in Fv-null FLC recipients were only mildly decreased, the FV activity within the platelet compartment was reduced to less than 1% of that in normal mice. We conclude that the murine platelet FV compartment is derived exclusively from primary biosynthesis within cells of marrow origin, presumably megakaryocytes, and that an intact platelet FV pool is not required for protection from spontaneous hemorrhage or bleeding following minor trauma.