Extracellular vesicles (EVs) harvested from cell culture supernatants of human mesenchymal stromal cells (MSCs) suppress acute inflammation in preclinical models of various diseases. Furthermore, they promote regeneration of damaged tissues. Following successful clinical treatment of a steroid-refractory Graft-versus-Host-Disease (GvHD) patient with EVs prepared from conditioned media of human bone marrow (BM)-derived MSCs, we aim to improve MSC-EV production and quality control towards clinical application. Observing functional differences of independent MSC-EV preparations in vitro, we established an optimized murine GvHD model for the analysis of independent MSC-EV preparations in vivo. To this end, T cell depleted allogeneic BM cells co-transplanted with naive allogeneic spleen-derived T cells induced GvHD symptoms with reproducible strengths in mice being preconditioned by ionizing irradiation. Administration of MSC-EV preparations with confirmed in vitro immune modulatory properties at three consecutive days significantly suppressed GvHD symptoms. In contrast, application of MSC-EV preparations lacking these in vitro immune modulating capabilities failed to suppress GvHD symptoms. Thus, our results reveal therapeutic differences among independent MSC-EV preparations that had been produced in a standardized manner. Thus, given this functional heterogeneity, any individual MSC-EV preparation considered for the clinical application should be evaluated for its potency prior to administration to patients.