Bone marrow mesenchymal stromal cells (BM-MSCs) are a rare population of cells that gives rise to skeletal tissues and the hematopoietic stroma in vivo. Recently, we have demonstrated that BM-MSCs fulfill stringent in vivo stem cell criteria when propagated as non-adherent mesenspheres but not as adherent-cultured cells. Motivated by these profound functional differences, the current study aimed to identify potential important MSC regulators by investigating global gene expression profiles of adherent and non-adherent culture-derived BM-MSCs in comparison with primary BM-MSCs. A substantial number of genes were differentially expressed between primary and culture-expanded cells already early upon culture, and numerous genes were found to be different when comparing adherent and non-adherent BM-MSCs. Cluster analysis identified 16 sets of genes of which two displayed comparable gene expression levels in primary and non-adherent cultured cells, but not in adherent cultured cells. This pattern suggested that these clusters contained candidate regulators of BM-MSCs. Gene expression differences were confirmed for selected genes and BM-MSC transcription factors by protein analysis and RT-PCR, respectively. Taken together, these data demonstrated profound gene expression changes upon culture of primary BM-MSCs. Moreover, gene cluster differences provide the basis to uncover the regulatory mechanisms that control primary and cultured BM-MSCs.