Skeletal muscle injury may occur from repetitive short bursts of biomechanical strain that impair muscle function. Alternatively, variations of biomechanical strain such as those held for long-duration are used by clinicians to repair muscle and restore its function. Fibroblasts embedded within the unifying connective tissue of skeletal muscle experience these multiple and diverse mechanical stimuli and respond by secreting cytokines. Cytokines direct all stages of muscle regeneration including myoblasts differentiation, fusion to form myotubes, and myotube functionality. To examine how fibroblasts respond to variations in mechanical strain that may affect juxtapose muscle, a myofascial junction was bioengineered that examined the interaction between the two cell types. Fibroblasts were experimentally shown to increase myoblast differentiation, and fibroblast biomechanical strain mediated the extent to which differentiation occurred. Intereleukin-6 is a strain-regulated cytokine secreted by fibroblasts was determined to be necessary for fibroblast-mediated myoblast differentiation. Myotubes differentiated in the presence of strained fibroblasts express greater number of acetylcholine receptors, greater acetylcholine receptor sizes, and modified to be more or less sensitive to acetylcholine-induced contraction. This study provides direct evidence that strained and non-strained fibroblasts can serve as a vehicle to modify myoblast differentiation and myotube functionality. Further understanding the mechanisms regulating these processes may lead to clinical interventions that include strain-activated cellular therapies and bioengineered cell engraftment for mediating the regeneration and function of muscle in vivo.