The gene regulatory networks that regulate heart formation include several layers spanning extracellular signaling pathways to DNA regulatory elements. We have established a set of approaches that includes mouse and induced pluripotent stem (iPS) cell models of congenital heart disease (CHD), to comprehensively understand the molecular basis of chamber formation, and the pathways that go awry in CHD. First, an allelic series of the transcription factor-encoding gene Tbx5 provides us with a range of CHDs spanning ventricular septation defects to single ventricle morphology. Using single cell RNA sequencing and single cell chromatin accessibility assays we are defining the gene regulatory networks that depend on TBX5. By comparing to a human iPS model of TBX5 haploinsufficiency we can leverage the power of the combined approaches. Finally, we have used machine-learning algorithms to predict alterations in genome folding in patients with single ventricle disease that harbor genetic copy variations. Overall these avenues are bringing clear mechanisms and testable hypotheses to understand the molecular underpinnings of single ventricle disease.