Cardiovascular disease is the major cause of death in the United States, with 80 million people suffering from some form of heart disease each year. One major limitation is the inability of the heart to repair the damaged tissue. Stem cell therapy holds enormous promise to repair and regenerate the damaged myocardium, but there are many technical difficulties that must first be overcome. One such difficulty is the present lack of ability to track and assess transplanted stem cells over time in vivo. The central hypothesis of this thesis is that in vivo bioluminescence imaging is a safe and useful method for monitoring transplanted stem cells in mouse hearts. To evaluate this hypothesis, two aims were performed. In aim 1, stable recombinant mES cell lines expressing the firefly luciferase (fLUC) reporter gene under the control of constitutive and cardiac-specific promoters were generated and characterized in vitro. In aim 2, these fLUC-expressing recombinant cell lines were evaluated following transplantation into neonatal mouse hearts. The major findings are: (1) Novel stable recombinant mES reporter cell lines were developed for in vivo bioluminescence imaging; (2) One of these cell lines was created using the glyceraldehyde 3-phosphodehydrogenase (GAPDH) promoter fused to the fLUC reporter and it showed similar levels of fLUC expression in undifferentiated (pluripotent) compared to cardiac-differentiated mES cells; (3) Another cell line was produced using the cardiac-specific sodium-calcium exchanger 1 (NCX1) promoter fused to the fLUC reporter and this cell line showed markedly increased fLUC expression following induction of cardiac differentiation in culture when compared to the pluripotent cells. (4) Transplantation of the recombinant fLUC-expressing cells into neonatal mouse hearts produced bioluminescent signals that persisted for at least 24 days, the maximum timepoint analyzed in this study; (5) Transplantation of 100,000 or more mES cells to the heart consistently produced teratoma and tumor formations, regardless of which recombinant clone was used or whether the mES cells were injected as pluripotent or cardiac-differentiated cells, (6) Transplantation of between 10,000 and 50,000 cardiac-differentiated NCX1-fLUC mES cells(containing mixed population of other cells) per heart resulted in measurable bioluminescent image signals in vivo with low incidence of tumor formation, and (7) Some of the transplanted NCX1-fLUC mES cells were identified in ventricular muscle tissue in postmortem histological sections where it was found that they had developed cardiomyocyte characteristics. In summary, I developed stable recombinant mES cell lines suitable for non-invasive bioluminescence imaging to study the survival and proliferation of the cells in vivo. These results demonstrate that bioluminescence imaging in the neonatal mouse heart model is an effective strategy for non-invasive monitoring of transplanted stem cells over time in vivo, and minimizes animal usage through elimination of the need for animal sacrifice at multiple timepoints.