In the past decade, mesenchymal stromal cells (MSCs) have developed a substantial portfolio in clinical trial investigations, becoming the focus of many regenerative immunotherapies. Besides being multipotent and having self-renewal capacity, MSCs possess immunosuppressive and anti-inflammatory properties. It is this immunosuppressive capacity of MSCs that has been highlighted in many preclinical studies for the treatment of various unrelated conditions. However, the mechanisms underlying their immunosuppressive effects remain poorly understood. We showed that upon intravenous (i.v.) administration, MSCs localise to the lungs and undergo caspase 3-dependent apoptosis within 1 hour. Furthermore, efferocytosis of apoptotic MSCs modulated the effector functions of lung phagocytes, especially alveolar macrophages. Secondary lymphoid organs, such as the spleen, are responsible for trapping foreign antigens entering via bloodstream and peripheral tissues. Therefore, we expected to see the spleen as central to the establishment of anti-inflammatory effects seen in MSC therapy. In an OVA-induced allergic asthma model, the immunosuppressive effects of MSC administration were abrogated in splenectomised mice. MSCs failed to inhibit lung eosinophilia and OVA-specific T cell restimulation responsiveness, including IL-5 and IL-13 production, in splenectomised mice. Splenectomised mice treated with MSCs had comparable levels of mucous production to their untreated counterparts, evident through lung histology. Overall, the spleen is indispensable for the immunosuppressive effects of i.v.-injected MSCs in OVA allergic asthma. Our data warrant a deeper investigation into the mechanistic role of splenic immune cell populations in MSC therapy.