AbstractTissue engineering utilizing periodontal ligament stem cells (PDLSCs) has recently been proposed for the development of new periodontal regenerative therapies. Although the use of autologous PDLSC transplantation eliminates the potential of a significant host immune response against the donor cells, it is often difficult to generate enough PDLSCs from one donor source due to the variation of stem cell potential between donors and disease state of each patient. In this study, we examined the immunomodulatory properties of PDLSCs as candidates for new allogeneic stem cell‐based therapies. Human PDLSCs displayed cell surface marker characteristics and differentiation potential similar to bone marrow stromal stem cells (BMSSCs) and dental pulp stem cells (DPSCs). PDLSCs, BMSSCs, and DPSCs inhibited peripheral blood mononuclear cell (PBMNC) proliferation stimulated with mitogen or in an allogeneic mixed lymphocyte reaction (MLR). Interestingly, gingival fibroblasts (GFs) also suppressed allogeneic PBMNC proliferation under both assay conditions. PDLSCs, BMSSCs, DPSCs, and GFs exhibited non‐cell contact dependent suppression of PBMNC proliferation in co‐cultures using transwells. Furthermore, conditioned media (CM) derived from each cell type pretreated with IFN‐γ partially suppressed PBMNC proliferation when compared to CMs without IFN‐γ stimulation. In all of these mesenchymal cell types cultured with activated PBMNCs, the expression of TGF‐β1, hepatocyte growth factor (HGF) and indoleamine 2, 3‐dioxygenase (IDO) was upregulated while IDO expression was upregulated following stimulation with IFN‐γ. These results suggest that PDLSCs, BMSSCs, DPSCs, and GFs possess immunosuppressive properties mediated, in part, by soluble factors, produced by activated PBMNCs. J. Cell. Physiol. 219: 667–676, 2009. © 2009 Wiley‐Liss, Inc.