The survival of thick artificial tissue in vivo requires the formation of three dimensional vessel networks that can anastomose with host vasculature and transport blood to the central part of the tissue quickly after implantation. In this study, a prevascularization strategy was used to create vessel networks in the engineered tissue constructs. The prevascularized tissue model was developed by co-culture of human umbilical vein endothelial cells (HUVECs) and fibroblasts in a fibrin gel. Interconnected vessel networks with visible lumens were developed within the tissue construct in 1 week. In order to demonstrate whether the preformed vessel networks can be functional in vivo, the prevascularized tissue as well as unprevascularized controls were implanted subcutaneously on the dorsal surface of immune-deficient mice. HUVEC-lined vessels containing red blood cells were evident in the prevascularized tissue by day 5, significantly earlier than non-prevascularized tissues (14 days). In addition, collagen deposition and larger number of proliferating cells were observed in the prevascularized tissue indicating higher level of cell viability. In order to further reduce the time required for the formation of functional anstomosis, the seeding density of HUVECs and fibroblasts were optimized. The development of vessel networks in vitro and the formation of functional anastomosis in vivo were significantly accelerated at the presence of high density of fibroblasts (2 million/ml) compared to low density of fibroblasts (0.2 million/ml). Finally, HUVECs were replaced by the endothelial cells derived from cord blood endothelial progenitor cells (EPC-ECs). Vessels formed with EPC-ECs showed similar kinetics as HUVECs in vitro. However, implantation of the prevascularized tissue construct in mice revealed a dramatic difference in the ability of EPC-ECs and HUVECs to form anastomosis with the host vasculature. Vascular beds derived from EPC-ECs were perfused within one day of implantation whereas no HUVEC vessels were perfused at day 1. Furthermore, while almost 90% of EPC-EC derived vessel networks were perfused at day 3, only one third of HUVEC derived vascular beds were perfused. These results demonstrated that prevascularization of engineered tissue constructs with well developed vessel network is an effective strategy to achieve fast perfusion of the engineered tissue constructs in vivo after implantation. This method will potentially facilitate the development of large three dimensional engineered tissues.