Vessel wall-derived somatic endothelial colony-forming progenitor cells (ECFCs) are key players in vascular homeostasis and regeneration. Due to their robust proliferative potential and profound vessel-forming capacity, ECFCs are considered to represent an attractive tool for vascular regenerative medicine and a promising target for antiangiogenic tumor therapy. Here, we describe an easily applicable method for isolating ECFCs directly from unmanipulated adult human blood and an animal protein-free large-scale expansion system to generate more than 100 million functional ECFCs.