Background: Mesenchymal stem cells (MSCs) are proven to have immunosuppressive functions in vitro and in vivo via various mechanisms. We aim to demonstrate these mechanisms by treating GVHD murine models with human MSCs. Methods: Balb/c host mice were subjected to irradiation and received C57BL/6N donor T cell depleted bone marrow cells only (negative control) or with donor spleen CD4+ T lymphocyte (positive control). 12 mice were given 1 x 10ˆ6 human MSCs via tail vein (treatment group). 2 host mice received Dil-stained MSCs. Survival rate was monitored. At day 6 post HSCT, single cell suspensions, tissue lysates and paraffin sections were prepared from target tissues of 4-6 host mice for (1) flow cytometry to check for the presence of donor T lymphocytes in various tissues, (2) ELISA to check for levels of proinflammatory cytokines, chemokine ligands and T cell cycle regulatory pathway testing, (3) western blotting for expression of inducible nitric oxide synthase (iNOS) and (4) imaging by coherent anti-stokes raman spectroscopy (CARS). Tissues from 2 host mice who received Dil-stained MSCs were prepared for in vivo imaging studies at day 6 post HSCT. 2 mice sacrificed for histopathologic observation are given 3 doses of MSC and sacrificed at day 50 post HSCT. Results and conclusion: The immunosuppressive functions of MSCs were documented in our GVHD models by (1) Protection of GVHD mice from death probably due to decrease donor T lymphocytes in target tissues and levels of TNF-a, IFN-g and RANTES expression, (2) Increased levels of iNOS, an immunosuppressive soluble mediator, (3) Suppression of phosphorylation of STAT 5A/B proteins, indicative of T cell cycle arrest, (4) Less severe GVHD pathologic changes in the lungs, liver and colon; and (5) Documentation of the presence of MSCs in target tissues by observing increased fluorescence signals via in vivo imaging studies and differences in chemical bond vibratory signals using CARS.