Microvascular endothelial cells display a large degree of heterogeneity in function depending on their location in the vascular tree. The existence of organ-specific, microvascular-bed-specific, and even intravascular variations in endothelial cell gene expression emphasizes their high cell-to-cell variability, which is furthermore extremely adaptable to altering conditions. The ability of microvascular endothelial cells to respond dynamically to pathology-related microenvironmental changes is particularly apparent in tumor-growth-associated angiogenesis. An understanding of how they behave, how their behavior varies between and within tumors, and how their behavior is related to responsiveness to drugs is critical for the development of effective anti-angiogenic treatment strategies. In this review, we introduce some general issues concerning organ-imprinted microvascular heterogeneity and highlight the importance of studying microvascular endothelial cell behavior in an in vivo context. This is followed by an overview of state-of-the-art knowledge regarding the nature of the variation in microenvironmental conditions in pre-clinical and clinical tumors and their consequences for tumor endothelial behavior. We provide recent insights into the in vivo molecular activation status of the endothelium and, finally, outline our current understanding of the way that anti-angiogenic drugs affect tumor endothelial cells in relation to their anti-tumor effects.