Previous clinical trials have shown that mesenchymal stromal cells (MSCs) can modulate graft versus host disease (GvHD) after allogeneic hematopoietic transplantation, although only moderate therapeutic effects have been observed in clinical trials. With the aim of increasing the anti-GvHD therapeutic effect of these cells, adipose tissue derived human MSCs (Ad-MSCs) were transduced with a bicistronic lentiviral vector, conferring stable expression of CXCR4, a molecule involved in cell migration, and IL-10, a cytokine with potent anti-inflammatory properties. Genetically modified MSCs (CXCR4-IL10-MSCs) showed the characteristic phenotype of MSCs, and no changes in the characteristic cytokine secretion profile were observed, except in IL10 secretion. In vitro experiments showed that the overexpression of these molecules in Ad-MSCs, efficiently enhanced their migration towards SDF-1α and improved their immunomodulatory properties compared to unmodified Ad-MSCs (WT-MSCs). Additionally, the efficacy of CXCR4-IL10-MSCs was increased in a xenogeneic GvHD mouse model generated by the infusion of human peripheral blood mononuclear cells into immunodeficient NSG mice, observing a significant decrease in the severity of the clinical and histological signs of the disease, compared to WT-MSC treated mice. Moreover, CXCR4-IL10-MSCs reduced the proliferation of pro-inflammatory Th1 and Th17 cells and induced T cells polarization towards an anti-inflammatory profile (CD3+-IL10+ cells) and increased the number of regulatory T and B cells, leading to a reduced disease impact in the target organs of GvHD. Taken together, our preclinical studies strongly suggest that CXCR4-IL10-MSCs should constitute an important new generation of MSCs for the treatment of GvHD in patients transplanted with allogeneic hematopoietic grafts.