Volumetric muscle loss and muscle degeneration are conditions for which there are currently no effective treatment options. Human adipose stem cells (hASCs) offer promise in cell-based regenerative therapies to treat muscle damage due to their ability to self-renew and differentiate. However, in the absence of universal culture conditions that yield greater than 15% myogenic differentiation, the clinical potential of these cells is limited. Here we report on the evaluation of two different media recipes, three extracellular matrix (ECM) proteins, and a poly (ethylene glycol) (PEGDMA) hydrogel with a physio-logically relevant elasticity to determine how the extracellular chemical and physical environment work together to enhance myogenic differentiation of hASCs. Our results identify a combination of unique biochemical and physical factors that promote myogenesis, laying the groundwork for creating a scaffold and culture medium that will effectively and efficiently direct myogenic differentiation of adult stem cells for clinical applications in the future. (C) 2020 Elsevier Inc. All rights reserved.