The heart is the first organ to form during development; however, little is known about the mechanisms that control the initial stages of cardiac differentiation. To investigate this process, we used a protein kinase expression screen, in which nonbeating embryonic stem (ES) cells were compared with beating ES cell-derived cardiomyocytes. We found that JAK2 experienced a 70% increase in protein levels within beating areas. Inhibition of JAK2 pharmacologically or by using dominant/negative JAK2 both resulted in diminished beating within embryoid bodies (EBs), whereas gain of function analysis using dominant/positive JAK2 resulted in a significant induction of beating. More important, inhibition of STAT3, a specific target of JAK2, by dominant/negative STAT3 resulted in the virtual complete loss of beating areas. Reverse transcription-polymerase chain reaction and Western analysis of STAT3-inhibited EBs resulted in lack of expression of several cardiac-specific genes, many of which contain within their promoter STAT3 DNA-binding regions. Taken together, the data reveal that the JAK2/STAT3 pathway is essential for initial stages of cardiomyogenesis.