Tissue cells generally pull on their matrix attachments and balance a quasi-static contractility against adequate adhesion, but any correlation with and/or influence on phenotype are not yet understood. Here, we begin to demonstrate how differentiation state couples to actomyosin-based contractility through adhesion and substrate compliance. Myotubes are differentiated from myoblasts on collagen-patterned coverslips that allow linear fusion but prevent classic myotube branching. Postfusion, myotubes adhere to the micro-strips but lock into a stress fiber-rich state and do not differentiate significantly further. In contrast, myotubes grown on top of such cells do progress through differentiation, exhibiting actomyosin striations within one week. A compliant adhesion to these lower cells is suggested to couple to contractility and accommodate the reorganization needed for upper cell striation. Contractility is assessed in these adherent cells by mechanically detaching one end of the myotubes. All myotubes, whether striated or not, shorten with an exponential decay. The cell-on-cell myotubes relax more, which implies a greater contractile stress. The non-muscle myosin II inhibitor blebbistatin inhibits relaxation for either case. Myotubes in culture are thus clearly prestressed by myosin II, and this contractility couples to substrate compliance and ultimately influences actomyosin striation.