Background. We previously reported the in vitro spontaneous transformation of human mesenchymal stem cells (MSC) generating a population with tumorigenic potential, that we termed transformed mesenchymal cells (TMC).Methodology/Principal Findings. Here we have characterized the molecular changes associated with TMC generation. Using microarrays techniques we identified a set of altered pathways and a greater number of downregulated than upregulated genes during MSC transformation, in part due to the expression of many untranslated RNAs in MSC. Microarray results were validated by qRT-PCR and protein detection.Conclusions/Significance. In our model, the transformation process takes place through two sequential steps; first MSC bypass senescence by upregulating c-myc and repressing p16 levels. The cells then bypass cell crisis with acquisition of telomerase activity, Ink4a/Arf locus deletion and Rb hyperphosphorylation. Other transformation-associated changes include modulation of mitochondrial metabolism, DNA damage-repair proteins and cell cycle regulators. In this work we have characterized the molecular mechanisms implicated in TMC generation and we propose a two-stage model by which a human MSC becomes a tumor cell.DR and SG received predoctoral fellowships from the Spanish Ministry of Education and Science, JG-C and L L-F received postdoctoral fellowships from the Ministry of Science and Technology and the Ministerio de Sanidad y Consumo (FIS; CP03/0031 and CP06/0267). This work was partially supported by Spanish Ministry of Science and Technology (CICYT) grants SAF2001-2262, SAF2005-0864 and GEN2001-4856-C13-02 to AB. The Department of Immunology and Oncology was founded and is supported by the Spanish National Research Council (CSIC) and by Pfizer.Peer reviewed