Isolation of mesenchymal stromal cells (MSCs) from the umbilical cord blood (UCB) has a success rate of 25% and is frequently contaminated by osteoclast-like cells (OLCs). CD271 is a well-known marker for the enrichment of bone marrow (BM) MSCs. We have assessed the effect of CD271 isolation on the isolation rate of MSCs from UCB. Twenty-one samples of UCB were collected. Ten samples of UCB and five of BM underwent CD271 isolation using magnetic activated cell sorting. The other 11 UCB samples were used as the control. The isolated cells were cultured and MSC isolation was confirmed with respect to morphology, flow cytometry, adipogenic and osteogenic differentiation potentials. CD271-positive UCB cells did not show outgrowth despite 54.5% MSCs isolation in the non-enriched portion. No OLC was noted in the CD271-enriched group, but 66% of the non-enriched samples were contaminated. All the CD271-positive BM cells formed MSC colonies. Although the per cent of CD271+ cells showed no difference between BM-mononuclear cells (MNCs) and UCB-MNCs, the haematopoietic marker, CD45, was found in a higher percentage of CD271-positive UCB-MNCs. The results of our study indicate that, although CD271 is a valuable marker for enrichment of MSCs from BM, it does not contribute to isolation of MSCs from UCB. In this source, most of the CD271+ cells are from haematopoietic origin, and possibly the process of isolation may eliminate the very low frequent MSCs and the isolation therefore fails.