Mutations in the NSD1 gene, which encodes the histone methyltransferase nuclear receptor binding set domain protein 1 (NSD1), cause a rare overgrowth condition known as Sotos syndrome. Despite more than 700 unique mutations having been reported in patients, most of these mutations converge on a common phenotype of megalencephaly/macrocephaly accompanied by mild to severe intellectual disability. Animal models are unable to recapitulate clinical features observed in humans and are thus inadequate for studying the underlying disease mechanisms. Therefore, to investigate how mutations in the NSD1 gene lead to Sotos syndrome, we have generated brain organoids from patient-derived induced pluripotent stem cell (iPSC) lines and their isogenic controls which we gene-corrected using CRISPR/Cas9 thus, establishing a significant resource for investigating Sotos syndrome. Western blot and real-time quantitative PCR analyses indicate that in patient-derived iPSC lines, NSD1 protein levels are significantly reduced compared to controls, while mRNA levels remain unchanged, suggesting that the mutations disrupt the post-transcriptional regulation of the NSD1 gene. Immunohistochemical profiling of the patient iPSC-derived brain organoids reveals increased levels of proliferation and neural progenitor markers suggesting that the NSD1 mutations affect early brain development by potentially altering neurogenesis. Additionally, by leveraging single-cell multi-omic approach and electrophysiology assays with the iPSC-derived brain organoids, we are investigating the epigenomic, transcriptomic, and functional changes caused by NSD1 mutations during brain development. This project will contribute to elucidate the role of NSD1 in the pathogenesis of Sotos syndrome, paving the way for potential therapeutic strategies.