Human umbilical cord blood (CB) is a potential source for mesenchymal stem cells (MSC) capable of forming specific tissues, for example, bone, cartilage, or muscle. However, difficulty isolating MSC from CB (CB-MSC) has impeded their clinical application. Using more than 450 CB units donated to two public CB banks, we found that successful cell recovery fits a hyper-exponential function of time since birth with very high fidelity. Additionally, significant improvement in the isolation of CB-MSC was achieved by selecting cord blood units having a volume ≥90 ml and time ≤2 h after donor's birth. This resulted in 90% success in isolation of CB-MSC by density gradient purification and without a requirement for immunoaffinity methods as previously reported. Using MSC isolated from bone marrow (BM-MSC) and adipose tissue (AT-MSC) as reference controls, we observed that CB-MSC exhibited a higher proliferation rate and expanded to the order of the 1 × 109 cells required for cell therapies. CB-MSC showed karyotype stability after prolonged expansion. Functionally, CB-MSC could be more readily induced to differentiate into chondrocytes than could BM-MSC and AT-MSC. CB-MSC showed immunosuppressive activity equal to that of BM-MSC and AT-MSC. Collectively, our data indicate that viable CB-MSC could be obtained consistently and that CB should be reconsidered as a practical source of MSC for cell therapy and regenerative medicine using the well established CB banking system. J. Cell. Biochem. 112: 1206–1218, 2011. © 2011 Wiley-Liss, Inc.