TBX5 is a transcription factor which plays important roles in the development of the heart and upper-limbs. Mutations in this gene produce the inherited disorder Holt-Oram syndrome. Here we report a physical interaction between TBX5 and MEF2C leading to a synergistic activation of alpha cardiac myosin heavy chain (MYH6). Mutants of TBX5, TBX5G80R and TBX5R279X that produce severe cardiac phenotypes impair the synergy. Using FRET, we demonstrate the interaction of TBX5 and MEF2C in living cells. We also show that they physically associate through their DNA-binding domains to form a complex on the MYH6 promoter. Morpholino- mediated knockdown of Tbx5 and Mef2c in zebrafish suggests that the genetic interaction of these proteins is not only required for MYH6 expression, but also is essential for the early stages of heart development and survival. This is the first report of a functional interaction between a T-box protein and a MADS-box factor that may be crucial in cardiomyocyte differentiation.