Cardiac progenitor cells of the second heart field contribute to growth of the heart tube during looping morphogenesis and ultimately give rise to a large part of the definitive heart, including outflow tract, right ventricular and atrial myocardium. Perturbation of SHF deployment results in a spectrum of congenital heart defects, in particular affecting conotruncal and atrial septal development. We have previously demonstrated that the transcription factor encoding gene Tbx1, a critical regulator of second heart field development and major gene underlying DiGeorge syndrome in man, is required for development of a specific subpopulation of progenitor cells that give rise to myocardium at the base of the pulmonary trunk. Failure of this subpulmonary myocardial domain is thought to be the primary defect underlying a spectrum of common conotruncal anomalies, including tetralogy of Fallot. Failure of development of this region of the heart in Tbx1 mutant mouse embryos is associated with coronary artery patterning defects. Here we present evidence for abnormal cell behavior in the second heart field of Tbx1 null embryos providing new insights into how defects in early second heart field development impact on later steps of cardiac morphogenesis. In addition, we characterize of genes specifically expressed in future subpulmonary myocardium identified from a microarray screen and initial data integrating these genes into a Tbx1 dependent gene regulatory network operating in this clinically relevant subpopulation of the second heart field.