Skeletal muscle regeneration is essential in conditions like muscle injury and muscular dystrophy, primarily relying on the activation and differentiation of myogenic progenitors. This study explores the role of redox-modulated surface thiol modification in myogenesis, using tris(2-carboxyethyl)phosphine hydrochloride (TCEP) to reduce disulfide bonds and generate free thiol groups on the cell surface. The findings show that TCEP treatment significantly increases cell surface thiols, activating the focal adhesion kinase (FAK)-phosphoinositide 3-kinase (PI3K)-protein kinase B (AKT) signaling pathway, and promoting cell spreading, adhesion, and actin cytoskeleton remodeling. This biochemical modulation upregulates myogenic and fusion-related markers, facilitating multinucleated myotube formation. Biophysical analysis using traction force microscopy (TFM), intracellular force microscopy (IFM), and monolayer stress microscopy (MSM) demonstrates that TCEP-treated cells, particularly at muscle-relevant stiffness (20 kPa), exhibit increased traction, intracellular, and monolayer stresses compared to untreated cells. These results suggest that redox-modulated surface thiol modification, combined with specific stiffness conditions, enhances myogenic differentiation and myotube maturation through biomechanical signaling. This proof-of-concept study advances the understanding of how redox-modulated surface engineering and substrate stiffness regulate myogenesis, offering novel therapeutic strategies for muscle regeneration and optimization of stem cell-based therapies for muscle tissue repair.