Abstract Body: Background: Down syndrome (DS) is the most common autosomal aneuploidy 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. Although DS-associated CHD is commonly attributed to chromosome 21 gene-dosage effects, human model systems are needed to pinpoint how trisomy 21 disrupts cardiac lineage decisions and downstream cardiomyocyte function. Objective: Identify defects in cardiac lineage commitment in DS+AVSD iPSC-derived cardiomyocytes (iPSC-CMs), assess 3D cardioid morphogenesis, and identify functional phenotypes. Methods: Patient iPSCs were generated from a DS+AVSD proband and a sex-matched unaffected family control and differentiated using a Wnt activation/inhibition cardiomyocyte protocol. Single-cell RNAseq was performed at Days 5, 10, 14 and 30. For each stage, analyses included differential gene expression, pathway enrichment, pseudotime reconstruction (Monocle), intercellular signaling (CellChat), and gene regulatory network analysis (SCENIC). Day 30 iPSC-CMs were further characterized by immunostaining for ventricular markers, extracellular flux measurements of mitochondrial respiration, and calcium imaging to assess beating behavior and electrical coupling. 3D Cardioids were imaged at media changes to monitor growth and collected at Days 2, 5, 10, and 20 for immunostaining. Results: At Day 5, DS cells demonstrated enrichment of gene expression programs associated with myogenesis and contractile development while at Days 10–14, DS cardiac progenitors displayed emerging mitochondrial and RNA-splicing stress pathways. By Day 30, DS iPSC-CMs exhibited reduced expression of conduction-related pathways, diminished connexin-43 accumulation at intercellular junctions, and disorganized excitation–contraction machinery, consistent with perturbed calcium cycling and impaired mitochondrial function. In parallel, DS-derived 3D cardioids modeling left ventricular and atrioventricular canal regions exhibited proliferation differences and cavity size discrepancies with chamber-marker expression delays. Conclusion: Patient-specific DS iPSC-CMs and 3D cardioids reproduce key cardiac abnormalities relevant to DS-associated CHD and provide a platform for resolving cell-specific mechanisms driving these defects.