Purpose: Transplantation of autologous and/or allogeneic blood vessels is the most convenient treatment for vascular diseases. With regard to extensive need for blood vessels, developments in vascular tissue engineering are contributing greatly. In this study, our aim is to create intact small-diameter tubular vascular grafts cultivated in pulsatile flow bioreactor. Materials and Methods: CD146+ cell-based small-diameter vascular grafts were fabricated with ECM/glycosaminoglycans and polyurethane nanofibers. Characterization of the vascular graft was performed by SEM and WST-1. To mimic blood circulation in the bioreactor, human CD34+ cells cultured in megakaryocytes/platelets medium; then these cells were transferred inside of the vascular graft to mimic blood circulation. Cell differentiation was evaluated by flow cytometry and colony assay. Wright-Giemsa staining and polyploidy analysis were performed to show the differentiated cell population inside of the vascular graft. Anti-thrombogenic properties of the blood vessel were demonstrated by IF. Results: Polyurethane nanofibers provided a suitable environment for Human umbilical cord vein endothelial cells (HUVECs), and no significant cytotoxic effect was observed. Scanning electron microscopy (SEM) analysis of the tubular graft showed that under perfusion HUVECs, smooth muscle cells (SMCs) and fibroblasts formed layers that aligned on each other, respectively. The vascular graft was strong with a tensile strength of 0.70 MPa and elastic modulus of 0.007 GPa. When cultured in a bioreactor system, platelet adhesion to the vascular graft was remarkably low. Conclusion: In conclusion, this vascular graft may hold the potential to regenerate functional small-diameter vessels for cardiovascular tissue repair.