The myogenic basic helix-loop-helix (bHLH) and MEF2 transcription factors are expressed in the myotome of developing somites and cooperatively activate skeletal muscle gene expression. The bHLH protein Twist is expressed throughout the epithelial somite and is subsequently excluded from the myotome. Ectopically expressed mouse Twist (Mtwist) was shown to inhibit myogenesis by blocking DNA binding by MyoD, by titrating E proteins, and by inhibiting trans-activation by MEF2. For inhibition of MEF2, Mtwist required heterodimerization with E proteins and an intact basic domain and carboxyl-terminus. Thus, Mtwist inhibits both families of myogenic regulators and may regulate myotome formation temporally or spatially.