During the last decade it has been demonstrated that multipotent mesenchymal progenitors are present and can be isolated also from cord blood (CB). Recently, we managed to set up a standard protocol allowing the isolation of high proliferative potential mesenchymal stromal cells (MSCs) with multiple differentiation capabilities, whereas the generation rate of MSC-initiating colonies could still be further improved. Herein, we strikingly succeeded in defining some simple and basic culture conditions based on the use of a chemically defined medium that increased the colony isolation efficiency up to almost 80% of processed CB units. Importantly, this result was achieved irrespective of CB unit white blood cell content and time elapsed from delivery, two limiting parameters in the CBMSC field. Thus, this high efficiency is guaranteed without strict selection of the starting material. In addition, since we are profoundly concerned about how different culture conditions can influence cell behavior, we devoted part of this study to a deep characterization of the established CBMSC populations to confirm their stemness features in this novel isolation and culture system. Therefore, an extended study of their immunophenotype, including classical pericytic markers, and a detailed molecular analysis addressing telomere length and also stemness-related microRNA contribution were performed. In summary, we propose a straightforward, extremely efficient, and reliable approach to isolate and expand thoroughly characterized CBMSCs, even when poor-quality CB units are the only disposable source or there is no space for an isolation to fail.