Biological and physical factors interact to modulate blood response in a wounded vessel, resulting in a hemostatic clot or an occlusive thrombus. Wall shear stress (τw) and pressure differential (ΔP) across the wound from the lumen to the extravascular compartment may impact hemostasis. This thesis describes the design of a microfluidic device capable of flowing human blood over a side channel plugged with collagen (± tissue factor) while independently controlling ΔP and τw. Using this device we were able to investigate the impact of physiologic hemodynamics on the growth and architecture of human blood clots. Our results revealed that both wall shear rate and the transthrombus pressure gradient govern clot development leading to the formation of two distinct intrathrombus regions; a core of highly-activated platelets and fibrin covered by a shell of less-activated platelets. These regions mimic the activation gradients of clots formed in vivo. We demonstrated that core development was dependent on the transthrombus pressure gradient restricting thrombin localization while shell development was dependent on wall shear rates. We also found that fibrin polymerization inhibited thrombin activity at both arterial and venous shear rates. However, the mechanism of this inhibition is shear dependent. At venous shears thrombin activity is inhibited by γ'-fibrin(ogen) binding. While at arterial and pathological shear rates the clot forms a more dense structure leading to physical trapping of thrombin independent of γ'-fibrin(ogen) binding. Taken together our data supports a model where clot architecture is maintained under various conditions by shear-specific thrombin inhibition mechanisms. Lastly, we demonstrated that the prevailing hemodynamics dilute ADP and thromboxane to regulate platelet contractility, a newly defined flow sensing mechanism to regulate clot function. The field of in vitro hemostasis and thrombosis research has lacked an assay capable of independently studying the effects of ΔP and local τw on clot development and function. Our microfluidic device bridges this gap, while providing new insights into the mechanisms of hemostasis and thrombosis, where hemostatic clot development must balance both thrombotic and hemorrhagic risks in order to rapidly and controllably cease bleeding.