EPCs (Endothelial progenitor cells) are the precursors of endothelial cells that form the inner lining of blood vessels and are involved in neovascularization and re-endothelializaton of injured tissues. These cells have the potential for treating a multitude of angiogenic disorders giving a new hope in the arena of vascular biology. In the present study, we have demonstrated that cord blood derived expanded CD34 + cells can be differentiated into functional EPCs in vitro. Differentiation was performed using three different media conditions: media with a combination of various cytokines, media devoid of cytokines and a commercial medium. The differentiated cells were characterized based on their morphology as well as at the molecular and cellular level using RT-PCR, FACS and IC. Functionality of the differentiated cells was also analyzed by various assays such as dye uptake, lectin binding and in vitro tube formation. In brief, the results showed that EPCs cultured in the in-house media devoid of cytokines exhibited both CFU-ECs (Colony forming unitendothelial cells) and ECFCs (Endothelial colony forming cells). These differentiated cells expressed endothelial markers such as CD34, CD105, VEGFR2, VEGFR3, vWF, ecNOS and CD31 at a higher rate as compared to the other media studied, were also functional and secreted growth factors such as VEGF and bFGF. Thus, in this study, we have tried to derive an economically feasible and a scalable approach in successfully differentiating expanded CD34 + cells from cord blood to functional EPCs. However, the safety and efficacy of the differentiated cells needs to be determined in suitable preclinical animal models and further clinical studies.