Abstract Body: Background: Down syndrome (DS) is the most common genetic abnormality caused by an extra copy of chromosome 21 with nearly half of these individuals having a congenital heart defect (CHD), specifically an atrioventricular septal defect (AVSD), as a co-occurring condition. Current paradigm holds that CHD occurrence is due to the gene dosage imbalance caused by the triplication of chromosome 21. While mouse models have been developed to study DS and recapitulate some of the cardiac phenotypes present in the clinic, patient-specific induced pluripotent stem cells (iPSCs) provide a patient-specific context to investigate cardiac cellular differentiation deficits. Objective: Determine cardiac cell lineage commitment defects in DS+AVSD iPSC-CM and assess ventricular cardiomyocyte characteristics and functions. Methods: iPSCs were generated from the proband and gender-matched family member followed by a Wnt-ON/Wnt-OFF cardiomyocyte differentiation protocol. Single-cell RNAseq was performed on differentiating iPSCs at Days 5 (cardiac mesoderm), Day 10 (cardiac progenitors), Day 14 (early cardiomyocytes) and Day 30 (cardiomyocytes) of the differentiation process. Bioinformatic analysis included differential gene expression, pathway analysis, pseudotime trajectory, and cell-cell communication inference. Day 30 iPSC-derived cardiomyocytes (iPSC-CMs) were further assessed with immunofluorescence for ventricular markers, extracellular flux assay for mitochondrial respiration, and calcium imaging for beat frequency and cell-cell coupling. Results: Day 5 pathway analysis in DS versus Control showed a global increase in pathways associated with muscle cell development and muscle contraction while a decrease in mesoderm formation, GPCR signaling, and axis specification pathways. Day 10 and 14 DS cell populations show an emergence of mitochondrial and splicing dysfunction pathways in cardiac progenitors while endoderm progenitors showed an upregulation in inflammatory and cytokine pathways. D30 iPSC-CMs show significant downregulation in cardiac conduction pathways evidenced in vitro by decreased localization of gap junction protein, connexin-43, to cell-cell boundaries and abnormal formation of excitation-contraction proteins. Day 30 iPSC-CMs also exhibit aberrant calcium handling and mitochondrial dysfunction. Conclusion: DS iPSC-CMs recapitulate cardiac phenotypes seen in DS patients and provide a cell-type specific context to DS-related congenital heart defects.