Cell fate decisions of pluripotent embryonic stem (ES) cells are dictated by activation and repression of lineage-specific genes. We have found that two muscle-specific microRNAs, miR-1 and miR-133, promote mesoderm formation from human ES cells but have opposite functions in further differentiation into cardiac muscle progenitors. Furthermore, miR-1 and miR-133 were potent repressors of non-muscle gene expression and cell fate during mouse and human ES cell differentiation and functioned in part by repressing Notch signaling. Regulation of the timing of each miRNA’s action allows for regulating sequential steps during cardiac differentiation and more efficient generation of cardiac cells from human ES cells. Many additional targets of these miRNAs have been discovered and may regulate cardiac cell fate. These findings indicate that miRNAs may have general utility in regulating cell fate decisions from pluripotent ES cells and could be used for generating cardiomyocytes for regenerative purposes and for disease modeling in iPS cells.