Abstract Background Complex immunophenotypic repertoires defining discrete adipose-derived stem cell (ASC) subpopulations may hold a key toward identifying predictors of clinical utility. To this end, we sorted out of the freshly established ASCs four subpopulations (SPs) according to a specific pattern of co-expression of six surface markers, the CD34, CD73, CD90, CD105, CD146, and CD271, using polychromatic flow cytometry. Method Using flow cytometry-associated cell sorting and analysis, gating parameters were set to select for a CD73⁺CD90⁺CD105⁺ phenotype plus one of the four following combinations, CD34⁻CD146⁻CD271⁻ (SP1), CD34⁻CD146⁺CD271⁻ (SP2), CD34⁺CD146⁺CD271⁻ (SP3), and CD34⁻CD146⁺CD271⁺ (SP4). The SPs were expanded 700- to 1000-fold, and their surface repertoire, trilineage differentiation, and clonogenic potential, and the capacity to support wound healing were assayed. Results Upon culturing, the co-expression of major epitopes, the CD73, CD90, and CD105 was maintained, while regarding the minor markers, all SPs reverted to resemble the pre-sorted population with CD34⁻CD146⁻CD271⁻ and CD34⁻CD146⁺CD271⁻ representing the most prevalent combinations, followed by less frequent CD34⁺CD146⁻CD271⁻ and CD34⁺CD146⁺CD271⁻ variants. There was no difference in the efficiency of adipo-, osteo-, or chondrogenesis by cytochemistry and real-time RT-PCR or the CFU capacity between the individual SPs, however, the SP2^{CD73+90+105+34-146+271-} outperformed others in terms of wound healing. Conclusions Our study shows that ASCs upon culturing inherently maintain a stable distribution of immunophenotype variants, which may potentially disguise specific functional properties of particular downstream lines. Furthermore, the outlined approach suggests a paradigm whereby discrete subpopulations could be identified to provide for a therapeutically most relevant cell product.