In the United States, the need for cardiac cells for transplantation or research applications is immense. While over 300,000 individuals per year seek heart transplants, less than 2% will receive hearts. Human cardiomyocytes are also needed to investigate the safety of new chemical entities being developed as drug candidates because cardiac toxicity is a major cause of drug development failure. Despite the need for human cardiac cells, progress to provide human cardiac cells has been challenging. One approach is to utilize human pluripotent stem cells to generate large numbers of cardiomyocytes for transplantation purposes or for use in drug discovery and development work. Currently, however, there are few methods to produce large numbers of human cardiomyocytes on a biotechnology scale to address the need. The hypothesis of our work is that small molecules could be identified that direct human pluripotent stem cell differentiation to cardiomyocytes. The long term goal of this work is to create a toolbox of small molecule reagents useful to direct human stem cell differentiation into cardiomyocytes and thus address the need for human cardiac cells. Therefore, we screened a collection of about 550 pathway modulators in a human embryonic stem cell-based assay that probes cardiomyocyte differentiation. One compound from the screen, (i.e., IWR-1), a very potent “hit” was selected for further medicinal chemical refinement as a Wnt/β-catenin antagonist and as an agent that directs human stem cell differentiation into cardiomyocytes. In an iterative process of chemical synthesis and parallel evaluation for Wnt/β-catenin inhibition and cardiomyogenesis, several potent agents with improved pharmacodynamic and kinetic properties were elaborated. Cardiomyogenesis was verified by quantifying the effects of transcription factors expressed in human stem cells upon differentiation (i.e., Nkx2.5, αMHC, alpha activin) by RT-qPCR, image analysis and for selected compounds, flow cytometry. The results showed that a distinct Wnt/β-catenin inhibition structure-activity relationship emerged from the "Dynamic Medicinal Chemistry" approach. Potent Wnt/β-catenin antagonists that emerged from this optimization campaign were useful toolbox reagents to produce large numbers of human cardiomyocytes from human stem cells. The conclusion is that small molecules can be used to afford human cardiomyocyte differentiation from pluripotent stem cells. Application of this approach to human induced pluripotent stem (iPS) cells could afford cardiomyocytes that could be used in drug discovery and development applications (i.e., drug toxicity testing) as well as provide a new way to address “Disease in a Dish” issues and create cells for therapeutic utility.