In order to enhance the therapeutic potential, it is important that sufficient knowledge regarding the dynamic changes of adipose-derived stem cell (ASC) immunophenotypical and biological properties during in vitro growth is available. Consequently, we embarked on a study to follow the evolution of highly defined cell subsets from three unrelated donors in the course of eight passages on tissue culture polystyrene. The co-expression patterns were defined by panels encompassing seven and five cell surface markers, including CD34, CD146, CD166, CD200, CD248, CD271, and CD274 and CD29, CD31, CD36, CD201, and Stro-1, respectively. The analysis was performed using multichromatic flow cytometry. We observed a major paradigm shift, where the CD166-CD34+ combination which was found across all cell subsets early in the culture was replaced by the CD166+ phenotype as the population homogeneity increased with time. At all analysis points, the cultures were dominated by a few major clones that were highly prevalent in most of the donors. The selection process resulted in two predominant clones in the larger panel (CD166+CD34−CD146−CD271− CD274−CD248−CD200− and CD166+CD34+ CD146−CD271−CD274−CD248−CD200−) and one clone in the smaller panel (CD29+CD201+CD36− Stro-1− CD31−). The minor subsets, including CD166+CD34−CD146−CD271+CD274−CD248−CD200− and CD166+CD34+CD146+CD271−CD274−CD248−CD200−, and CD29+CD201−CD36−Stro-1−CD31−, CD29+CD201+CD36−Stro-1+CD31−, and CD29+CD201+CD36+Stro-1−CD31−, in the seven and five marker panels, respectively, were, on the other, hand highly fluctuating and donor-dependent. The results demonstrate that only a limited number of phenotypical repertoires are possible in ASC cultures. Marked differences in their relative occurrence between distinct individuals underscore the need for potency standardization of different ASC preparation to improve the clinical outcome.