The therapeutic efficacy of multipotent mesenchymal stromal cells (MSCs) is attributed to particular MSC-derived cytokines and growth factors. As MSCs are applied locally to target organs or home there after systemic administration, they experience diverse microenvironments that are biochemically and biophysically distinct. Here we use well-defined in vitro conditions to study the impact of substrate elasticity on MSC-derived trophic factors. By varying hydrogel compliance, the elasticity of brain and muscle tissue was mimicked. We screened >90 secreted factors at the protein level, finding a diverse elasticity-dependent expression pattern. In particular, IL-8 was up-regulated as much as 90-fold in MSCs cultured for 2 days on hard substrates, whereas levels were consistently low on soft substrates. In summary, we show substrate elasticity directly affects MSC paracrine expression, a relevant finding for therapies administering MSCs in vivo.