To engineer tissues with clinically relevant dimensions, one must overcome the challenge of rapidly creating functional blood vessels to supply cells with oxygen and nutrients and to remove waste products. We tested the hypothesis that endothelial cells, cocultured with osteoprogenitor cells, can organize into a prevascular network in vitro. When cultured in a spheroid coculture model with human mesenchymal stem cells, human umbilical vein endothelial cells (HUVECs) form a 3-dimensional prevascular network within 10 days of in vitro culture. The formation of the prevascular network was promoted by seeding 2% or fewer HUVECs. Moreover, the addition of endothelial cells resulted in a 4-fold upregulation of the osteogenic marker alkaline phosphatase. The addition of mouse embryonic fibroblasts did not result in stabilization of the prevascular network. Upon implantation, the prevascular network developed further and structures including lumen could be seen regularly. However, anastomosis with the host vasculature was limited. We conclude that endothelial cells are able to form a 3-dimensional (3D) prevascular network in vitro in a bone tissue engineering setting. This finding is a strong indication that in vitro prevascularization is a promising strategy to improve implant vascularization in bone tissue engineering.