Gene overlap serves as a common strategy employed by bacteria and viruses to expand their genomic capacity. Over the past two decades, advances in omics studies have unveiled a prevalence of overlapped genes in eukaryotes, including mammals. However, few are reported to participate in the regulation of the cardiovascular system. In this study, we introduce a novel protein-coding nested gene, named Aff3ir, which contributes to endothelial maintenance by promoting the differentiation of vascular stem/progenitor cells (SPCs) into endothelial cells (ECs).