Mesenchymal stromal cells (MSCs) are one of the most frequently used cell sources for tissue engineering strategies. Cultivation of osteogenic MSCs is a prerequisite for cell-based concepts that aim at bone regeneration. Quantitative real time reverse transcription polymerase chain reaction (qRT-PCR) analysis is a commonly used method for the examination of mRNA expression levels. However, data on suitable reference genes for osteogenically cultivated MSCs is scarce. Hence, the aim of the study was to compare the regulation of different potential reference genes in osteogenically stimulated MSCs. Human MSCs were isolated from bone marrow aspirates of N=6 hematologically healthy individuals, expanded by polystyrene-adherence, and maintained with and without osteogenic supplements for 14 days. Cellular proliferation and osteogenic differentiation were assessed by total DNA quantification, cell-specific alkaline phosphatase (ALP) activity and by qualitative staining for ALP and alizarin red, respectively. mRNA expression levels of N=32 potential reference genes were quantified using the human Endogenous Control TaqMan® assays. mRNA expression stability was calculated using geNorm. The combined use of the most stable reference genes and DNA-damage-inducible alpha, Pumilio homolog 1, and large ribosomal protein P0 significantly improved gene expression accuracy as compared to the use of the commonly used reference genes beta actin and glyceraldehyde-3-phosphate dehydrogenase during qRT-PCR-based target gene expression analysis of osteogenically stimulated MSCs.