AbstractBackground  GDNF/RET and Endothelin‐3 (ET‐3)/EDNRB regulate survival, differentiation, migration, and proliferation of neural crest‐derived cells. Although several RET and EDNRB signalling mediators have been characterized, most of the genes targeted by these two pathways are still largely unknown. We focused our study on apolipoprotein B (APOB) as a novel target gene of the RET and EDNRB pathways, based on previous data obtained using a Caenorhabditis elegans strain mutant for the homologue of mammalian ECE1.Methods  Molecular and cellular studies of Apob were performed in the murine Neuro2a cells, an in vitro model for studying neural crest‐derived cell development, along with a mouse knock‐in for the Hirschsprung‐associated mutation RetC620R. Silencing for Apob and Ret has been performed via shRNA.Key Results  GDNF/RET and ET‐3/EDNRB cooperated in inducing neuronal differentiation resulting in Apob activation in Neuro2a cell line. Apob expression was downregulated in mouse embryos homozygous for the RetC620R mutation and presenting a severe Hirschsprung phenotype. Ret silencing prevented Apob expression increase. MAPK P38 kinase activation evoked Apob expression via GDNF/RET signalling in Neuro2a cells. A p53‐dependent repressor element in Apob promoter resulted in a reduced Apob expression. Silencing of Apob reduced HuD protein expression.Conclusions & Inferences  Apob is a novel downstream target of the RET/EDNRB pathways with a role in neuronal survival and maintenance, as indicated by its effect on HuD expression. Our data provide a conceptual framework to investigate and establish the role of APOB gene in severe gut dysmotility.