NONO deficiency in hiPSCs results in a distinct defect in early cardiomyocyte differentiation. Mechanistically, NONO interacts with HOXA1 and regulates the dynamic expression of key genes during early cardiomyocyte differentiation. ChIP-seq analysis reveals that NONO loss reduces HOXA1 occupancy at target genes, compromising its transcriptional regulation. Additionally, NONO and HOXA1 cooperatively activate the Wnt signaling.