Background: Umbilical cord blood (UCB) is an easily accessible alternative source for multipotent mesenchymal stromal cells (MSCs) and is generally believed to provide MSCs with a higher proliferative potential compared with adult bone marrow. Limitations in cell number and strict dependence of expansion procedures from selected lots of fetal bovine serum have hampered the progress of clinical applications with UCB-derived MSCs. Methods: We analyzed the isolation and proliferative potential of human UCB MSCs compared with bone marrow MSCs under optimized ex vivo culture conditions. We further investigated human platelet lysate as an alternative to replace fetal bovine serum for clinical-scale MSC expansion. Clonogenicity was determined in colony-forming units-fibroblast assays. MSC functions were tested in hematopoiesis support, vascular-like network formation and immune modulation potency assays. Results: MSCs could be propagated from UCB with and without fetal bovine serum. MSC propagation was effective in 46% of UCB samples. Once established, the proliferation kinetics of UCB MSCs did not differ significantly from that of bone marrow MSCs under optimized culture conditions, resulting in more than 50 population doublings after 15 weeks. A clinical quantity of 100 million MSCs with retained differentiation potential could be obtained from UCB MSCs within approximately 7 weeks. Ex vivo expansion of hematopoietic UCB-derived CD34⁺ cells as well as immune inhibition and vascular-like network formation could be shown for UCB MSCs propagated under both culture conditions. Conclusion: We demonstrate for the first time that human MSCs can be obtained and propagated to a clinical quantity from UCB in a completely bovine serum-free system. Surprisingly, our data argue against a generally superior proliferative potential of UCB MSCs. Functional data indicate the applicability of clinical-grade UCB MSCs propagated with human platelet lysate-conditioned medium for hematopoiesis support, immune regulation and vascular regeneration.