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Abstract Stimuli–responsive biomaterials may be used to better control the release of bioactive molecules or cells for applications involving drug delivery and controlled cell release. In this study, we developed a Factor Xa (FXa)‐responsive biomaterial capable of controlled release of pharmaceutical agents and cells from in vitro culture. FXa‐cleavable substrates were formed as hydrogels that degraded in response to FXa enzyme over several hours. Hydrogels were shown to release both heparin and a model protein in response to FXa. Additionally, RGD‐functionalized FXa‐degradable hydrogels were used to culture mesenchymal stromal cells (MSCs), enabling FXa‐mediated cell dissociation from hydrogels in a manner that preserved multicellular structures. Harvesting MSCs using FXa‐mediated dissociation did not influence their differentiation capacity or indoleamine 2,3‐dioxygenase (IDO) activity (a measure of immunomodulatory capacity). In all, this FXa‐degradable hydrogel is a novel responsive biomaterial system that may be used for on‐demand drug delivery, as well as for improving processes for in vitro culture of therapeutic cells.