The regulatory effect of human multipotent mesenchymal stromal cells (MSC) on allogenic T lymphocytes is extremely powerful and of important clinical relevance, but the mechanisms underlying this process are not fully elucidated. We report here that T lymphocytes activated with a sub-mitogenic stimulus such as phytohemaglutinin alone (PHA), or with mitogenic stimuli such as PHA + interleukin-2 (P-IL2), or immobilized anti-CD3 + anti-CD28 mAb (a3-28), tightly bound allogenic MSC and transmigrated within 4 h under them, where they remained for approximately 60 h. Allogenic MSC induced T cell proliferation in cultures containing sub-mitogenic PHA concentrations, and inhibited the mitogenic effect of P-IL2 or a3-28. Anti-gamma-IFN mAb or L-tryptophan complementation partially restored proliferation in P-IL2 and a3-28 cultures, whereby gamma-IFN-synthesizing CD3+ cells were detectable. MSC-lymphocyte contact hindrance using transwells abrogated proliferation in PHA cultures, restored it integrally in P-IL2 cultures, and partially in a3-28 cultures. These data suggest that MSC-induced T lymphocyte regulation results from the combination of various processes. Allogenic cell-cell contact, as demonstrated by the PHA co-cultures is per se stimulatory, whereas gamma-IFN synthesized by activated T lymphocytes, which activates indolamine 2,3-dioxygenase in MSC, and L-tryptophan depletion, which is induced by this enzyme, are inhibitory. Transmigration is nevertheless pivotal for the establishment of the inhibition by these mediators because it targets lymphocytes under the stroma in small extracellular spaces surrounded by MSC, where L-tryptophan is efficiently destroyed, leading to T lymphocyte proliferation arrest. In conclusion lymphocyte transmigration under allogenic MSC potentiates the inhibitory effect of soluble mediators generated by these cells.