For their clinical use Mesenchymal Stromal Cells (MSCs), isolated from bone marrow (BM-MSCs) are considered Advanced Therapy Medicinal Products (ATMP) and need to be produced according to Good Manufacturing Practice (GMP). Human platelet lysate (HPL) represents a good GMP-compliant alternative to animal serum and after pathogen inactivation with Psoralen was more efficient and safer to produce MSCs in GMP. In this study MSCs cultivated in FBS (FBS-MSC) or inactivated HPL (iHPL-MSC), were compared for their immunomodulant properties. In particular, the effects of MSCs on: 1)proliferation of total Lymphocytes (Ly) and on naïve T Ly subsets induced to differentiate versus Th1 and Th2 Ly; 2) the immunophenotype of different T cell subsets; 3)the cytokine release to verify Th1, Th2 and Th17 polarization were analyzed by using in vitro co-culture system. We observed that iHPL-MSCs showed the same immunomodulant properties observed in the FBS-MSCs co-cultures. Although, a more efficient effect on the increase of naïve T cells and, in the Th1 cytokine release related to iHPL was observed. This study confirms that iHPL, used as medium supplement, may be considered a good alternative to FBS for a GMP-compliant MSC expansion to preserve their immunomodulant proprieties.