Current treatment options are limited for patients with myocardial infarction or heart failure. Cellular cardiomyoplasty is a promising therapeutic strategy being investigated as a potential treatment, which aims to deliver exogenous cells to the infarcted heart, for the purpose of restoring healthy myocardial mass and mechanical cardiac function. While several cell types have been studied for this application, only bone marrow cells and human mesenchymal stem cells (hMSCs) have been shown to be safe and effective for improving cardiac function in clinical trials. In both human and animal studies, the delivery of hMSCs to infarcted myocardium decreased inflammatory response, promoted cardiomyocyte survival, and improved cardiac functional indices. While the benefits of using hMSCs as a cell therapy for cardiac repair are encouraging, the desired expectation of cardiomyoplasty is to increase cardiomyocyte content that will contribute to active cardiac mechanical function. Delivered cells may increase myocyte content by several different mechanisms such as differentiating to a cardiomyocyte lineage, secreting paracrine factors that increase native stem cell differentiation, or secreting factors that increase native myocyte proliferation. Considerable work suggests that hMSCs can differentiate towards a cardiomyocyte lineage based on measured milestones such as cardiac-specific marker expression, sarcomere formation, ion current propagation, and gap junction formation. However, current methods for cardiac differentiation of hMSCs have significant limitations. Current differentiation techniques are complicated and tedious, signaling pathways and mechanisms are largely unknown, and only a small percentage of hMSCs appear to exhibit cardiogenic traits. In this body of work, we developed a simple strategy to initiate cardiac differentiation of hMSCs in vitro. Incorporating environmental cues typically found in a myocardial infarct (e.g. decreased oxygen tension and increased concentrations of cell-signaling factors), our novel in vitro conditioning regimen combines reduced-O2 culture and hepatocyte growth factor (HGF) treatment. Reduced-O2 culturing of hMSCs has shown to enhance differentiation, tissue formation, and the release of cardioprotective signaling factors. HGF is a pleiotropic cytokine involved in several biological processes including developmental cardiomyogenesis, through its interaction with the tyrosine kinase receptor c-Met. We hypothesize that applying a combined conditioning treatment of reduced-O2 and HGF to hMSCs in vitro will enhance cardiac-specific gene and protein expression. Additionally, the transplantation of conditioned hMSCs into an in vivo infarct model will result in differentiation of delivered hMSCs and improved cardiac mechanical function. In testing our hypothesis, we show that reduced-O2 culturing can enhance hMSC growth kinetics and total c-Met expression. Combining reduced-O2 culturing with HGF treatment, hMSCs can be conditioned to express cardiac-specific genes and proteins in vitro. Using small-molecule inhibitors to target specific effector proteins in a proposed HGF/c-Met signaling pathway, treated reduced-O2/HGF hMSCs show a decrease in cardiac gene expression. When implanted into rat infarcts in vivo, reduced-O2/HGF conditioned hMSCs increase regional cardiac mechanics within the infarct region at 1 week and 1 month. Further analysis from the in vivo study showed a significant increase in the retention of reduced-O2/HGF conditioned hMSCs. Immunohistochemistry showed that some of the reduced-O2/HGF conditioned hMSCs express cardiac-specific proteins in vivo. These results suggest that a combined regimen of reduced-O2 and HGF conditioning increases cardiac-specific marker expression in hMSCs in vitro. In addition, the implantation of reduced-O2/HGF conditioned hMSCs into an infarct significantly improves cardiac function, with contributing factors of improved cell retention and possible increases in myocyte content. Overall, we developed a simple in vitro conditioning regimen to improve cardiac differentiation capabilities in hMSCs, in order to enhance the outcomes of using hMSCs as a cell therapy for the diseased heart.