Appropriate hemostasis hinges on normal vascular control of platelet function to suppress thrombosis in the absence of blood vessel injury. In this regard, normal intercellular communication between vascular endothelial cells and platelets is integral to maintaining platelets in an inactivated state and inhibiting maladaptive clot (thrombus) formation. Platelets interact with endothelial cells in a variety of ways and drive hemostatic or pathological phenotypes. Platelets can interact in contact-dependent or -independent fashion with the vessel wall and affect endogenous synthesis of nitric oxide, prostaglandin I2, endothelial cell surface-associated ecto-ADPase/CD39, and adhesion proteins. Analysis of differences in platelet activity with respect to different vascular beds and understanding the relevance of hypoxia, inflammation, blood flow, and oxidant stress to platelet adhesion, activation, and aggregation are essential for understanding the diverse pathological phenotypes of thrombosis. It is also anticipated that clarifying the biophysical and molecular sequence of events underpinning endothelial regulation of platelet activation will require next-generation methods that utilize whole organ platforms in silico to identify novel mediators of thrombosis in vivo and prevent the attendant adverse consequences in patients. Dissecting the molecular patterns responsible for regulation of platelet activity with respect to endothelial–platelet interactions is vital for modulating pathological thrombosis and achieving homeostasis.