Upon vessel injury, a series of events are orchestrated to promote an optimal hemostatic response that can stop blood loss and maintain homeostasis of the vascular system. Understanding this process in humans has been mainly limited to clinical observations and in vitro systems that do not take into account the dynamic environment in which the hemostatic events take place in vivo. This thesis aims to expand the deployment of novel microfluidic approaches that can recapitulate the different microenvironments to which blood is exposed at the injury site during hemostasis. With these platforms, we gained insights about the biochemical and biophysical cues that shape the hemostatic plug. First, we developed a novel microfluidic system that mimics the interface between the vascular wall and flowing human blood to model hemostasis upon injury. This platform allowed us to monitor the hemostatic response from vessel injury to hemostatic closure, achieved by a platelet- and fibrin-rich plug. We also showed the contribution of platelet-driven collagen matrix deformation to hemostatic closure. Furthermore, the proof-of-concept responsiveness to an antiplatelet and an anticoagulant therapy highlighted the potential use of this microfluidic system as a drug screening platform. Secondly, we studied the relationship between thrombin generation under flow with coagulation factors and platelets. Using a system developed by Dr. Scott Diamond’s group, we evaluated the impact of tissue factor, platelets, platelet aggregation, and FVIII deficiency to thrombin generation under flow. We found that, in this system, thrombin generation was fully dependent on tissue factor and platelets, but independent of platelet aggregation. We also found that FVIII deficiency decreased thrombin generation over a collagen/TF surface. The measured thrombin levels correlated with platelet activation and fibrin deposition, and with clinical measures of FVIII percentage and activated partial thromboplastin time. Overall, this thesis highlights the potential application of microfluidic systems in the field of hemostasis and thrombosis as complementary platforms in preclinical and clinical research to achieve a more comprehensive view of the multiple processes that work together to achieve hemostasis.