The high mobility group type A-2 (HMGA 2) transcription factor is involved in proliferation and differentiation, mainly during embryogenesis. Its activated form (HMGA 2/T) presents oncogenic activities both in vivo and in vitro. However, its precise role during embryogenesis is unknown. We investigated its role during the commitment of mouse embryonic stem (ES) cells by constructing cell lines expressing either wild type (wt) or HMGA 2/T forms of the gene. Following differentiation, control and wt HMGA 2 ES cells did not display myotubes; whereas HMGA 2/T ES cell lines massively formed contractile myotubes. Furthermore, as opposed to control cells, HMGA 2/T ES cells highly expressed the muscle myosin heavy chain (MHC) marker. Interestingly, in experimental conditions inhibitory for myogenesis, we observed a strong expression of MyoD and myogenin in HMGA 2/T cells. By contrast, commitment into adipocyte, neuron, and cardiomyocyte lineages was not affected. Teratocarcinomas induced by HMGA 2/T ES cell lines presented numerous skeletal muscle-differentiated tissues that were not observed in wt HMGA 2 or control tumours. Finally, rapamycin, an inhibitor of the mTOR kinase, downregulated endogenous HMGA-2 expression and inhibited myogenesis. This effect was prevented by overexpression of exogenous HMGA-2. Our results reveal a novel function of HMGA-2 in skeletal muscle differentiation.