Loss of cardiomyocytes through cardiac injury and disease substantially impairs cardiac function. Given that cardiomyocytes permanently exit the cell cycle soon after birth, the heart has traditionally been considered a terminally differentiated organ, and thus unable to effectively regenerate. However, recent discoveries have challenged this paradigm and have provided the foundation for exploring the prospect of inducing cardiomyocytes to reenter the cell cycle and, in turn, regenerate damaged myocardium. This has proven challenging, as mammalian cardiomyocytes appear to lose competency to respond robustly to pro-proliferative stimuli following a neonatal maturation period. During this maturation period, cardiomyocytes exit the cell cycle, undergo structural and metabolic changes, and most become binucleated. We and others have observed that the small percentage of mature cardiomyocytes that remain mononucleated (MoNuc) are more competent to respond to proliferative stimuli than their binucleated (BiNuc) counterparts and therefore offer a biological model with which to interrogate loss of proliferative competency in cardiomyocytes. We hypothesized that differences in the maturation processes of MoNucs and BiNucs leave them differentially primed to respond to proliferative stimuli. We devised a novel method to obtain populations highly enriched in MoNuc and BiNuc cardiomyocytes by fluorescence associated cell sorting (FACS) and profiled the transcriptional differences between MoNuc and BiNuc cardiomyocytes at three different timepoints spanning cardiomyocyte maturation. Transcriptome analysis revealed that during the neonatal period, binucleation is associated with the termination of a proliferation-associated gene expression program in exchange for a mature cardiomyocyte gene expression program. Our data also suggested that an E2f/Rb transcriptional network is central to the divergence of these two populations and that remnants of the differences acquired during the neonatal period remain in adult cardiomyocytes. Furthermore, we induced binucleation by genetically blocking the ability of cardiomyocytes to complete cytokinesis, which allowed us to directly link binucleation to changes in Rb/E2f signaling and loss of regenerative competency. The research presented in this dissertation suggests a potential mechanism by which the binucleation process may result in loss of regenerative capacity.