Thrombus formation following vascular injury is an essential component of both hemostasis and pathologic vessel occlusion. This process occurs in a closed, pressurized environment in which blood flows rapidly over the injury site. Thrombus formation must occur quickly to reduce blood loss, but is carefully modulated to limit vessel occlusion. Circulating cells, plasma proteins, vessel wall components, and physical forces such as shear all influence thrombus formation. Historically, thrombus formation has been studied by isolating the separate components of blood involved in clot formation. With improved optical techniques, investigators have increasingly studied thrombus formation under conditions of flow in vitro and in live animals in vivo. Using multichannel fluorescence intravital videomicroscopy, the authors have studied the changes in the kinetics and deposition of platelets, fibrin, and tissue factor at the injury site during thrombosis in transgenic mice, bone marrow transplanted mice, and mice treated with pharmacological agents that modulate thrombosis. The differences in the kinetics of accumulation of the various components of thrombus in these mice have provided new insights about thrombus formation in arterioles. This review discusses the role of platelet intracellular signaling, P-selectin expression on platelets, and tissue factor-bearing microparticles in thrombus formation.