BACKGROUND: Mesenchymal stem cells (MSCs) require priming by proinflammatory stimuli for optimal immunosuppressive effects. Our previous work identified mixed lymphocyte reaction-conditioned medium (MLR-CdM) as a potent enhancer of MSC immunosuppressive properties. This study evaluates the immunomodulatory potential of MSC-derived extracellular vesicles preconditioned with MLR-CdM (MSC-EVMLR) compared to IFN-γ (MSC-EVIFN), focusing on key miRNAs and mechanisms involved. METHODS: We assessed the ability of MSC-EVMLR and MSC-EVIFN to modulate lymphocyte proliferation and cytokine expression in vitro. To identify potential effector molecules within MSC-EVMLR, we performed miRNA array analysis combined with dose-response experiments using MLR-CdM under varying stimulation conditions. We used a murine allogeneic heterotopic heart transplantation model to investigate the impact of MSC-EVMLR on graft survival and its immunomodulatory effects. RESULTS: MSC-EVMLR outperformed MSC-EVIFN in suppressing lymphocyte proliferation and steering cytokine expression toward an anti-inflammatory profile in vitro. Through miRNA array analysis and dose-response experiments with MLR-CdM, miR-638 was identified as a potential effector molecule in MSC-EVMLR. In vivo study demonstrated that MSC-EVMLR significantly prolonged graft survival, which was associated with a marked decreased proinflammatory cytokines IL6 and IFN-γ and increase in regulatory T cells (Tregs) and within the transplanted heart tissue. These effect was significantly reduced upon miR-638 knockdown. Additionally, the miR-638/Fosb axis was identified as a key pathway that promoted Treg differentiation and induced immune tolerance. CONCLUSIONS: Preconditioning MSCs with MLR-CdM, a blend of inflammatory stimuli, potentiates the immunoregulatory capacity of MSC-EV beyond the effects of IFN-γ stimulation alone. This study advances the understanding of MSC-EV-based therapies in transplantation.