The vascular system forms the largest surface in our body, serving as a critical interface between blood circulation and our diverse organ/tissue environments. Thus, the vascular system performs a gatekeeper function for organ/tissue homeostasis and the body’s adjustment to pathological challenges. The endothelium, as the most inner layer of the vasculature, regulates the tissue microenvironment, which is critical for development, hemostatic balance, inflammation, and angiogenesis, with a role as well in tumor malignancy and metastasis. These multitudinous functions are primarily mediated by organ/tissue-specifically differentiated endothelial cells, in which heterogeneity has long been recognized at the molecular and histological level. Based on these general principles of vascular-bed heterogeneity and characterization, this review largely covers landmark discoveries regarding organ/tissue microenvironment-governed endothelial cell phenotypic changes. These involve the physical features of continuous, discontinuous, fenestrated, and sinusoidal endothelial cells, in addition to the more specialized endothelial cell layers of the lymphatic system, glomerulus, tumors, and the blood brain barrier (BBB). Major signal pathways of endothelial specification are outlined, including Notch as a key factor of tip/stalk- and arterial-endothelial cell differentiation. We also denote the shear stress sensing machinery used to convey blood flow-mediated biophysical forces that are indispensable to maintaining inert and mature endothelial phenotypes. Since our circulatory system is among the most fundamental and emergent targets of study in pharmacology from the viewpoint of drug metabolism and delivery, a better molecular understanding of organ vasculature-bed heterogeneity may lead to better strategies for novel vascular-targeted treatments to fight against hitherto intractable diseases.