Therapeutic benefit of stem cells has been demonstrated in multiple disease models and clinical trials. Robust quality assurance is imperative to make advancements in culturing procedures to enable large-scale cell manufacturing without hampering therapeutic potency. MicroRNAs (miRNAs or miRs) are shown to be master regulators of biological processes and are potentially ideal quality markers. We determined miRNA markers differentially expressed under nonclinical multipotent adult progenitor cell (MAPC) and mesenchymal stem cell (MSC) culturing conditions that regulate important stem cell features, such as proliferation and differentiation. These bone marrow-derived stem cell types were selected because they both exert therapeutic functions, but have different proliferative and regenerative capacities. To determine cell-specific marker miRNAs and assess their effects on stem cell qualities, a miRNA and mRNA profiling was performed on MAPCs and MSCs isolated from three shared donors. We applied an Ingenuity Pathway Analysis-based strategy that combined an integrated RNA profile analysis and a biological function analysis to determine the effects of miRNA-mRNA interactions on phenotype. This resulted in the identification of important miRNA markers linked to cell-cycle regulation and development, the most distinctive being MAPC marker miR-204-5p and MSC marker miR-335-5p, for which we provide in vitro validation of its function in differentiation and cell cycle regulation, respectively. Importantly, marker expression is maintained under xeno-free conditions and during bioreactor isolation and expansion of MAPC cultures. In conclusion, the identified biologically relevant miRNA markers can be used to monitor stem cell stability when implementing variations in culturing procedures. STEM CELLS TRANSLATIONAL MEDICINE 2016;5:709-722 SIGNIFICANCEHuman adult marrow stromal stem cells have shown great potential in addressing unmet health care needs. Quality assurance is imperative to make advancements in large-scale manufacturing procedures. MicroRNAs are master regulators of biological processes and potentially ideal quality markers. MicroRNA and mRNA profiling data of two human adult stem cell types were correlated to biological functions in silico. Doing this provided evidence that differentially expressed microRNAs are involved in regulating specific stem cell features. Furthermore, expression of a selected microRNA panel was maintained in next-generation culturing platforms, demonstrating the robustness of microRNA profiling in stem cell comparability testing.