Mesenchymal stromal cell (MSC)-based therapy holds great promise for treating immune disorders and for regenerative medicine in agreement with their paracrine trophic and immunosuppressive activities. Various processes have been developed worldwide to produce clinical grade MSCs but, so far, it is not known if one given MSC is more efficient than another. In addition, while their broad activity on innate and adaptative immune cell subsets is now widely admitted, the precise mechanisms supporting their immunoregulatory capacities are still a matter of debate. Finally, quantitative immunological potency assays correlated to clinical efficacy and clinically relevant immunomonitoring approaches for MSC-treated patients are sorely needed. Multiple parameters could influence the immunomodulatory potential of therapeutic MSCs. The most important challenge is now to differentiate, within a high number of poorly comparable and even contradictory pre-clinical studies, the parameters that could have some clinical impact from those that are only due to uncontrolled experimental variability. Importantly, besides MSC-related differences, primarily linked to production processes, several important variables associated with immune assays themselves, including selection of effector immune cells, activation signals, and read-out techniques, should be carefully considered to obtain solid results with potential therapeutic application. In this review, we establish a core of common and reproducible immunological properties of MSCs, shed light on technical issues concerning immunomodulatory potential assessment, and put them into perspective when considering clinical application.