Our recent study suggests that transplanted embryonic stem (ES) cells following myocardial infarction (MI) differentiate into the major cell types in the heart and improve cardiac function. However, the extent of regeneration was relatively meager compared to the observed functional improvement. The mechanisms underlying their improved function are completely unknown. In this report, we provide evidence using a cell culture model system for novel mechanisms that involve the release of cytoprotective, anti-apoptotic factor (s) from ES cells and inhibits H₂O₂ induced apoptosis in rat cardiomyocyte derived cell line H9c2. Conditioned medium (CM) from growing mouse ES cells treated with and without hydrogen peroxide (H₂O₂) was generated. Apoptosis was induced after exposure with H₂O₂ in H9c2 cells for two hours followed by replacement with fresh cell culture or ES cell-CM. After 24 hours, H9c2 cells treated with both ES cell-CMs demonstrated significant decrease in apoptosis as determined by TUNEL staining, apoptotic ELISA, caspase-3 activity and DNA ladder. Next, using luminex technology we examined presence of anti-apoptotic proteins cystatin c, osteopontin, clusterin and anti-fibrotic, tissue inhibitor metalloproteinase-1 (TIMP-1) in both ES cell-CMs and the level of released factors were 2-170 fold higher compared with H9c2 cell-CM. Anti-apoptotic effects of ES cell-CM were significantly inhibited with TIMP-1 antibody suggesting TIMP-1 is an important factor to inhibit apoptosis. Furthermore, we used CM from TIMP-1 over-expressing cell line and demonstrate that H₂O₂ induced apoptosis in the H9c2 cells were significantly inhibited. These observations demonstrate that factors released from ES cells contain anti-apoptotic factors and the effects are mediated by TIMP-1. Moreover, these findings suggest that released factors might be useful for therapeutic applications in the ischemic heart disease as well as for many other diseases.