Tissue factor pathway inhibitor (TFPI) is an alternatively spliced anticoagulant protein that primarily dampens the initiation phase of coagulation before thrombin is generated. As such, TFPI’s actions are localized to cells expressing TF and to sites of injury, where it is an important regulator of bleeding in hemophilia. The major splice isoforms TFPI? and TFPI? localize to different sites within and surrounding the vasculature. Both forms directly inhibit factor Xa (FXa) via their Kunitz 2 domain and inhibit TF-FVIIa via their Kunitz 1 domain in a tight complex primarily localized to cells. By forming complexes localized to distinct cellular microenvironments and engaging additional cell surface receptors, TFPI alters cellular trafficking and signaling pathways driven by coagulation proteases of the TF pathway. TFPI?, which circulates in complex with FV and protein S, also serves an inhibitor of FXa independent of the TF initiation complex and prevents the formation of an active prothrombinase. This regulation of thrombin generation in the context of vessel injury is effectively blocked by antibodies to Kunitz 2 domain of TFPI and exploited as a therapy to restore efficient hemostasis in hemophilia.