Mesenchymal stromal cells (MSCs) are used as salvage therapy to treat steroid-refractory acute graft-versus-host disease (aGvHD). We studied the immunological response to MSC treatment in 16 aGvHD patients by assessing lymphocyte profiles and three proposed aGvHD serum markers during the MSC treatment. Surprisingly, there were no obvious differences in the lymphocyte profiles between the responders and non-responders. The numbers of T, B, and NK cells were below the normal reference interval in all patients. CD4⁺ T helper (Th) cell levels remained particularly low throughout the follow-up period. The relative proportion of Th1 cells decreased, while regulatory T cells remained unaltered, and only very few Th2 and Th17 cells could be detected. Serum concentrations of regenerating islet-derived protein 3-alpha, cytokeratin-18 fragments (CK18F), and elafin were significantly elevated in patient samples compared with healthy controls, but only CK18F showed any potential in the prediction of patients’ response to MSCs. No obvious markers for MSC therapy response were revealed in this study, but the results suggest that allogeneic MSCs do not provoke overt T cell-mediated immune responses at least in immunosuppressed aGvHD patients. The results advocate for the safety of MSC therapy and bring new insights in MSC immunomodulation mechanisms.