Mesenchymal stem cells (MSC) robustly inhibit mitogen or alloantigen-driven T cell responses in vitro. The in vivo effectiveness of MSC as a monotherapy under inflammatory conditions however remains controversial. This study therefore aimed to identify novel ex vivo strategies to derive highly immunosuppressive MSC populations. Human bone-marrow derived MSC co-cultured with alloantigen or mitogen phytohaemagglutinin (PHA)-stimulated T cells showed baseline dose-dependent inhibition of T cell proliferation in a -thymidine incorporation assay. The exogenous addition of proinflammatory cytokines IFN- γ (100 U/ml) and IL-17A (50 ng/ml) in MSC-T cell co-cultures restored mitogen phytohaemagglutinin (PHA)-induced T cell proliferation while only IL-17A (5 ng/ml) showed reversal of MSC-inhibition on alloantigen-driven T cell responses. Interestingly, the 5 day preactivation of MSC with 500 U/ml IFN-γ (MSC-γ) or with 50 ng/ml IL-17A (MSC-17) derived a highly immunosuppressive population of cells compared to untreated-MSC (UT:MSC). In PHA assays, MSC-γ and MSC-17 inhibited T cell proliferation by 20.6 % (p=0.0120) and 37.6 % (p=0.0028) respectively. MSC-γ were more effective than MSC-17 at suppressing the alloantigen-driven T cell proliferation. Greater than 90 % MSC-γ expressed the T cell negative costimulatory molecule programmed death ligand-1 (PD-L1) demonstrated by flow cytometry. Neutralisation of PD-L1 in MSC-γ however failed to restore T cell proliferative responses suggesting a partial but non-exclusive role of PD-L1 in MSC-γ immunosuppression. MSC-17 on the other hand showed similar PD-L1 expression as UT:MSC. Furthermore, MSC-γ potentially induced T cell anergy evident by the reduction of T cell activation marker CD 25 by flow cytometry. Similar to UT:MSC, these proinflammatory cytokine modified MSC differentiated into adipocytes, osteocytes and chondrocytes. MSC-17 retained similar stem cell marker expression as UT:MSC. Although MSC-γ highly expressed MHC class II molecules, the absence of costimulatory molecule CD 80 and CD 83 expression indicate the inability of MSC-γ to function as T cell stimulators. Moreover, IFN-γ dose-dependently inhibited MSC growth but did not affect cell viability. On the contrary, IL-17A enhanced MSC proliferation compared to UT:MSC and MSC-γ. The enhancement of MSC growth potential implicates that MSC-17 are more beneficial to generate larger numbers of cells for in vivo infusion. In conclusion, MSC-γ and MSC-17 enhanced MSC immunosuppression on T cells. The modified cells also retained MSC stemness phenotype evident by the mesenchymal trilineage differentiation potential and the positive expression of stem cell markers. These ex vivo strategies to derived MSC-γ and MSC-17 could yield more potent cell therapy agents to ameliorate inflammatory responses and enable the MSC application as a monotherapy in vivo.