Mesenchymal stromal cell (MSC) apoptosis is required for in vivo immunosuppression. However, the induction of apoptosis is heavily dependent on the recipient's immune system. In graft-versus-host disease (GvHD), patients who fail to respond to MSCs are in fact those whose immune cells are unable to induce MSC apoptosis ex vivo. The information is critical to explain why responses in clinical trials vary even though the same sources of MSC products are infused. More importantly, it highlights the need for an alternative MSC treatment for the nonresponders. By using a mouse model of ovalbumin (OVA)-induced allergic inflammation, we demonstrated that we could generate apoptotic MSCs (ApoMSCs) in vitro and use them to successfully reduce allergic airway inflammation. In order to address the logistics of their potential future clinical application, we have shown that ApoMSCs could be cryopreserved without impairing efficacy compared to freshly generated ApoMSCs. We have also highlighted that MSCs need to undergo complete apoptosis before cryopreservation to retain their immunosuppressive activity. The cryopreserved ApoMSCs could serve as a potential future off-the-shelf cellular product, in particular for patients who suffer from inflammatory conditions yet do not harbor the immune capacity to induce MSC apoptosis in vivo. Our data provide proof-of-concept that under laboratory conditions, ApoMSCs can be successfully frozen and thawed without affecting their anti-inflammatory activity, as tested in a murine model of allergic inflammation.