Tissue factor pathway inhibitor (TFPI) blocks thrombin generation via the extrinsic blood coagulation pathway. Because the severe bleeding in patients with hemophilia occurs from deficiency of intrinsic blood coagulation pathway factor VIII or IX, pharmacological agents that inactivate TFPI and, therefore, restore thrombin generation via the extrinsic pathway, are being developed for treatment of hemophilia. Murine models of combined TFPI and factor VIII deficiency were used to examine the impact of TFPI deficiency on bleeding and clotting in hemophilia. In breeding studies, Factor VIII null (F8(-/-)) did not rescue the embryonic death of TFPI null (Tfpi(-/-)) mice. Tfpi(+/-) did not alter the bleeding phenotype of F8(-/-) mice. However, total inhibition of intravascular TFPI through injection of anti-TFPI antibody mitigated tail vein bleeding. Interestingly, tail blood loss progressively decreased at doses greater than needed to totally inhibit plasma TFPI, suggesting that inhibition of a sequestered pool of TFPI released at the injury site mitigates bleeding. Because TFPI is sequestered within platelets and released following their activation, the function of platelet TFPI was examined in F8(-/-) mice lacking hematopoietic cell TFPI that was generated by fetal liver transplantation. Blood loss after tail transection significantly decreased in Tfpi(+/-);F8(-/-) mice with hematopoietic Tfpi(-/-) cells compared with Tfpi(+/-);F8(-/-) mice with Tfpi(+/+) hematopoietic cells. Additionally, following femoral vein injury, Tfpi(+/-);F8(-/-) mice with Tfpi(-/-) hematopoietic cells had increased fibrin deposition compared with identical-genotype mice with Tfpi(+/+) hematopoietic cells. These findings implicate platelet TFPI as a primary physiological regulator of bleeding in hemophilia.