The multi-chambered mammalian heart arises from a simple tube by polar elongation, myocardial differentiation and morphogenesis. Members of the large family of T-box (Tbx) transcription factors have been identified as crucial players that act in distinct subprogrammes during cardiac regionalization. Tbx1 and Tbx18 ensure elongation of the cardiac tube at the anterior and posterior pole, respectively. Tbx1 acts in the pharyngeal mesoderm to maintain proliferation of mesenchymal precursor cells for formation of a myocardialized and septated outflow tract. Tbx18 is expressed in the sinus venosus region and is required for myocardialization of the caval veins and the sinoatrial node. Tbx5 and Tbx20 function in the early heart tube and independently activate the chamber myocardial gene programme, whereas Tbx2 and Tbx3 locally repress this programme to favour valvuloseptal and conduction system development. Here, we summarize that these T-box factors act in different molecular circuits and control target gene expression using diverse molecular strategies including binding to distinct protein interaction partners.