The clinical application of mesenchymal stromal/stem cells (MSCs) has been extensively explored. In this study, we examined the availability of freshly donated umbilical cord blood (UCB) units that do not qualify for the Japanese banking system for transplantation because of their small volume as a source of MSCs. Forty-five UCB units were used. The median volume of each UCB unit and number of nucleated cells per unit were 40 mL and 5.39 × 10(8), respectively. MSCs were successfully isolated from 18 of 45 units (40 %). The MSC isolation rate was not affected by cell processing method or the interval between delivery and cell processing. The volume of the UCB unit and the mononuclear cell count were predictive factors of the MSC isolation rate. MSCs were effectively isolated by selecting UCB units with a volume of ≥54 mL and containing ≥1.28 × 10(8) mononuclear cells, yielding a MSC isolation rate of >70 %. UCB-derived MSCs were similar to bone marrow-derived MSCs in terms of their morphology, surface marker expression, and differentiation potential, apart from adipogenesis. Our data indicate that UCB units that are currently discarded due to inadequate volume should be reconsidered as a source of MSCs using the well-established UCB banking system.