Preparations of mesenchymal stromal cells (MSCs) are generally obtained from unfractionated tissue cells, resulting in heterogeneous cell mixtures. Several markers were proposed to enrich these cells, but the majority of these markers are defined for bone marrow (BM). Moreover, the surface markers of freshly isolated MSCs also differ from those of cultured MSCs in addition to a phenotypic variation depending on the MSC source. For tissue engineering applications, it is crucial to start with a well-defined cell population. In this study, we performed immunomagnetic selections with five single surface markers to isolate MSC subpopulations from BM and adipose tissue (AT): CD271, SUSD2, MSCA-1, CD44, and CD34. We determined the phenotype, the clonogenicity, the proliferation, the differentiation capacity, and the immunoregulatory profile of the subpopulations obtained in comparison with unselected cells. We showed that native BM-MSCs can be enriched from the positive fractions of MSCA-1, SUSD2, and CD271 selections. In contrast, we observed that SUSD2 and MSCA-1 were unable to identify MSCs from AT, meaning they are not expressed in situ. Only the CD34(+) selection successfully isolated MSCs from AT. Interestingly, we observed that CD271 selection can define AT cell subsets with particular abilities, but only in lipoaspiration samples and not in abdominoplasty samples. Importantly, we found a population of clear CD34(+) fresh BM-MSCs displaying different properties. A single marker-based selection for MSC enrichment should be more advantageous for cell therapy and would enable the standardization of efficient and safe therapeutic intervention through the use of a well-identified and homogeneous cell population.