Myasthenia gravis (MG) is a chronic autoimmune disease characterized by autoantibodies targeting the neuromuscular junction and precluding normal nerve-to-muscle communication. Conventional treatments are limited by severe side effects mandating the set-up of novel therapies. Mesenchymal Stromal Cells (MSC) are multipotent progenitors presenting immunomodulatory capacities. Research grade (RG-) MSC conditioned with peripheral blood mononucleated cells (PBMC) improved clinical outcomes in our humanized MG mouse model (NSG-MG) (Sudres et al., JCI Insight, 2017). In clinical perspective, RG-MSC should be replaced by clinical grade (CG-) MSC. Here, we characterized and compared phenotypes, gene expression profiles and functional, in vitro and in vivo, capacities of resting and conditioned cells. Therefore, CG-MSC derived from adipose tissue were conditioned by PBMC (cMSC), left untreated (rMSC), or activated by IFN-γ (γ-MSC) as a control. Flow cytometry phenotyping showed that cMSC most remarkable phenotypic traits include increased CD54, CD273 and CD49a and reduced HLA-DR expression. At variance, IFNγ activation induced major changes in MSC phenotype in agreement with literature (increased CD54, HLA-ABC, HLA-DR, reduced CD59). Single cell clustering by mass cytometry suggested cMSC and rMSC proximity and underlined the phenotypic alterations induced by IFNγ. Gene expression study by RNA-Seq showed differential expression of 244 genes between rMSC and cMSC, while 2089 and 3614 genes were differentially expressed when comparing γ-MSC with rMSC and cMSC, respectively. Each conditioning involved particular pathways. In vitro immunomodulating capacities were evaluated by PBMC proliferation inhibition assays that showed that the cMSC supernatant reduced proliferation by at least 50%. Finally, cMSC were challenged in our NSG-MG mouse model, and cMSC-treated mice presented MG scores lowered by 50% compared to untreated mice from 2 weeks post-injection. To sum-up, this work unveiled treatment-dependent phenotypic markers of MSC and demonstrated that immunomodulation capacities in vitro and in vivo are enhanced by cellular conditioning