The velo-cardio-facial syndrome (VCFS)/DiGeorge syndrome (DGS) is a genetic disorder characterized by phenotypic abnormalities of the derivatives of the pharyngeal arches, including cardiac outflow tract defects. Neural crest cells play a major role in the development of the pharyngeal arches, and defects in these cells are likely responsible for the syndrome. Most patients are hemizygous for a 1.5- to 3.0-Mb region of 22q11, that is suspected to be critical for normal pharyngeal arch development. Mice hemizygous for a 1.5-Mb homologous region of chromosome 16 (Lgdel/+) exhibit conotruncal cardiac defects similar to those seen in affected VCFS/DGS patients. To investigate the role of Lgdel genes in neural crest development, we fate mapped neural crest cells in Lgdel/+ mice and we performed hemizygous neural crest-specific inactivation of Lgdel. Hemizygosity of the Lgdel region does not eliminate cardiac neural crest migration to the forming aortic arches. However, neural crest cells do not differentiate appropriately into smooth muscle in both fourth and sixth aortic arches and the affected aortic arch segments develop abnormally. Tissue-specific hemizygous inactivation of Lgdel genes in neural crest results in normal cardiovascular development. Based on our studies, we propose that Lgdel genes are required for the expression of soluble signals that regulate neural crest cell differentiation.