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Islet transplantation has the potential to cure type 1 diabetes, but current transplantation protocols are not optimal and there is extensive loss of islet β‐cell insulin secretory function during the immediate post‐transplantation period. Studies using experimental models of diabetes have shown that the coculture of islets with mesenchymal stromal cells (MSCs) prior to transplantation improves graft function, but several variables differed among research groups (e.g., type of MSCs used and the treatment conditions). We have therefore assessed the effects of MSCs on mouse and human islets by investigating the importance of tissue source for MSCs, the coculture protocol configuration and length, the effect of activated MSCs, and different β‐cell secretory stimuli. MSCs derived from adipose tissue (aMSCs) were the most effective at supporting β‐cell insulin secretion in both mouse and human islets, in a direct contact coculture configuration. Preculture with aMSCs enhanced both phases of glucose‐induced insulin secretion and further enhanced secretory responses to the non‐nutrients carbachol and arginine. These effects required a coculture period of 48–72 hours and were not dependent on activation of the MSCs. Thus, direct contact coculture with autologous, adipose‐derived MSCs for a minimum of 48 hours before implantation is likely to be an effective addition to human islet transplantation protocols. Stem Cells Translational Medicine 2019;8:935&944