Vascular endothelial cells (ECs), the inner layer of the blood vessels, are critical for vascular development, homeostasis maintenance, and pathological processes. ECs are subjected to hemodynamic shear stress due to their direct contact with blood, which regulates vascular homeostasis by modulating biological processes such as the development and cell fate of ECs. The cell fate transition of ECs mainly includes the generation of hematopoietic stem/progenitor cells that enter the circulatory system via endothelial-to-hematopoietic transition (EHT) and the formation of mesenchymal cells in vascular stenosis via endothelial-to-mesenchymal transition (End-MT) under pathological conditions. One common feature of both fate transitions is the disruption of intercellular adhesion. Here, we review the molecular mechanisms by which hemodynamic shear stress regulates EHT and EndMT in endothelial cells, providing valuable insights into the underlying mechanisms of cardiovascular development and related diseases.