Mesenchymal stem cells (MSCs) are multipotent progenitors with broad immunosuppressive properties. However, their therapeutic use in autoimmune disease models has shown dissimilar effects when applied at different stages of disease. We therefore investigated the effect of the addition of MSCs on the differentiation of Th1, Treg and Th17 cells in vitro, at different states of CD4+ T cell activation. CD4+ T lymphocytes purified by negative selection from mouse C57BL/6 splenocytes were cultured under Th1, Th17 and Treg inducing conditions with IL-12, TGF-β + IL-6 or TGF-β, respectively. C57BL/6 bone marrow derived MSCs were added to CD4+ T cell cultures at day 0 or after 3 days of T cell polarizing activation. Intracellular cytokines for Th1, Th17 and Treg cells were quantitated at day 6 by flow cytometry. While early addition (day 0) of MSCs suppressed all CD4+ T cell lineages, addition at day 3 only decreased IFN-γ production by Th1 polarized cells by 64% (p < 0.05) while markedly increased IL-17 production by Th17 polarized cells by 50% (p < 0.05) and left IL-10 production by Treg polarized cells unchanged. MSCs exhibit their typical suppressive phenotype when added early to cell cultures in the presence of CD4+ T cell polarizing stimuli. However, once T cell activation has occurred, MSCs show an opposite stimulating effect on Th17 cells, while leaving Treg IL-10 producing cells unchanged. These results suggest that the therapeutic use of MSCs in vivo might exert opposing effects on disease activity, according to the time of therapeutic application and the level of effector T cell activation.