Controlled matrix interactions were presented to pancreatic β-cells in three-dimensional culture within poly(ethylene glycol) hydrogels. Dispersed MIN6 β-cells were encapsulated in gel environments containing the following entrapped extracellular matrix (ECM) proteins: collagen type I, collagen type IV, fibrinogen, fibronectin, laminin, and vitronectin. In ECM-containing gels, β-cell survival was significantly better than in gels without ECM over 10 days. Correspondingly, apoptosis in encapsulated β-cells was less in the presence of each matrix protein, suggesting the ability of individual matrix interactions to prevent matrix signaling-related apoptosis (anoikis). MIN6 β-cells cultured in gels containing collagen type IV or laminin secreted more insulin in response to glucose stimulation than β-cells in all other experimental conditions. Variations in collagen type IV or laminin concentration between 10 μg/mL and 250 μg/mL did not affect insulin secretion. Finally, β-cell function in hydrogels presenting both collagen type IV and laminin revealed synergistic interactions. With a total protein concentration of 100 μg/mL, three gel compositions of varying ratios of collagen type IV to laminin (25:75, 50:50, and 75:25) were tested. In the presence of 25 μg/mL of collagen type IV and 75 μg/mL of laminin, β-cell insulin secretion was greater than with laminin or collagen type IV individually. These results demonstrate that specific, rationally designed extracellular environments promote isolated β-cell survival and function.