Umbilical cord blood (UCB) is recognized as an enriched source of endothelial progenitor cells (EPCs) with potential therapeutic value. Because cryopreservation is the only reliable method for long-term storage of UCB cells, the clinical application of EPCs depends on our ability to acquire them from cryopreserved samples; however, the feasibility of doing so remains unclear. In this study we demonstrate that EPCs can be isolated from cryopreserved UCB-derived mononuclear cells (MNCs). The number of outgrowth EPC colonies that emerged in culture from cryopreserved samples was similar to that obtained from fresh UCB. Furthermore, EPCs obtained from cryopreserved MNCs were phenotypically and functionally indistinguishable from freshly isolated ones, including the ability to form blood vessels in vivo. Our results eliminate the necessity of performing cell isolation procedures ahead of future clinical needs and suggest that EPCs derived from cryopreserved UCB may be suitable for EPC-related therapies.