AbstractThe phosphoinositide 3‐kinase (PI3K) family has multiple vascular functions, but the specific regulatory isoform supporting lymphangiogenesis remains unidentified. Here, we report that deletion of the Pik3r1 gene, encoding the regulatory subunits p85α, p55α, and p50α impairs lymphatic sprouting and maturation, and causes abnormal lymphatic morphology, without major impact on blood vessels. Pik3r1 deletion had the most severe consequences among gut and diaphragm lymphatics, which share the retroperitoneal anlage, initially suggesting that the Pik3r1 role in this vasculature is anlage‐dependent. However, whereas lymphatic sprouting toward the diaphragm was arrested, lymphatics invaded the gut, where remodeling and valve formation were impaired. Thus, cell‐origin fails to explain the phenotype. Only the gut showed lymphangiectasia, lymphatic up‐regulation of the transforming growth factor‐β co‐receptor endoglin, and reduced levels of mature vascular endothelial growth factor‐C protein. Our data suggest that Pik3r1 isoforms are required for distinct steps of embryonic lymphangiogenesis in different organ microenvironments, whereas they are largely dispensable for hemangiogenesis. Developmental Dynamics 238:2670–2679, 2009. © 2009 Wiley‐Liss, Inc.