Abstract Body (Do not enter title and authors here): Understanding the mechanisms of cardiomyocyte development is critical for fulfilling the potential of induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). Although myocyte development is known to depend on internal and external mechanical cues, further investigation is required to understand the contributions of different signals and how they are integrated together to generate an adult cardiomyocyte. Here, we address this gap by examining the role of calcium-activated contractility in sarcomere formation and maturation and its influence on the iPSC-CM response to nanopatterns. We generated iPSCs with homozygous D65A cardiac troponin C (cTnC) modifications. This engineered cTnC cannot bind to calcium at site II, resulting in tropomyosin blocking strong myosin binding to the thin filament and inhibiting sarcomere contraction. WT and D65A iPSCs were differentiated into cardiomyocytes and matured in culture over 60 days. Cells were characterized via fluorescence imaging and calcium transient analysis. WT and D65A proteomes were examined via mass spectrometry throughout differentiation and maturation. We also replated matured cardiomyocytes onto nanopatterned surfaces to investigate how external mechanical signals affect maturation in contractile versus non-contractile cells. Surprisingly, we found that sarcomeres formed in the cTnC D65A cardiomyocytes, though these sarcomeres were underdeveloped and disorganized. Cardiomyocytes with engineered cTnC D65A also exhibited significant proteomic maturation defects and abnormal calcium transients. Thus, calcium-activated contractility is dispensable for sarcomerogenesis but critical for cardiomyocyte maturation. Replating non-contractile, D65A cardiomyocytes onto nanopatterns improved myofibril structural organization and increased abundance of oxidative phosphorylation (OXPHOS) machinery. This suggests that nanopatterns enhance aspects of maturation in D65A myocytes and that external mechanical cues may partially compensate for defective contractility. In contrast, nanopatterns reduced sarcomeric and OXPHOS protein content in WT cardiomyocytes. Thus, nanopatterns did not facilitate and may have hindered WT maturation, potentially indicating that matured cardiomyocytes are not susceptible to nanopattern-mediated changes. In addition to these novel findings, these large mass spectrometry datasets cataloging iPSC-CM maturation represent a useful resource for the cardiovascular community.