The basic helix-loop-helix transcription factor Twist1 plays an essential role in mesenchymal cell populations during embryonic development and in pathological disease. Remodeling of the cardiac outflow tract (OFT) into the functionally separate aortic arch and pulmonary trunk is dependent upon the dynamic, coordinated contribution of multiple mesenchymal cell populations. Here, we report that Twist1(-/-) mice exhibit OFTs that contain amorphic cellular nodules within their OFT endocardial cushions. The nodular mesenchyme expresses the related bHLH factors Hand1 and Hand2, but reduced levels of the normal cushion marker Periostin. Lineage mapping confirms that nodule cells are exclusively of cardiac neural crest origin (cNCC), and are not ectopic cardiomyocytes or smooth muscle cells. These studies also reveal a delay in cNCC colonization of the OFT cushions. Furthermore, these mapping studies uncover nodules in the pharyngeal arches, and identify Twist1(-/-) neural crest cell defects within the dorsal neural tube, which exhibits an expanded domain of Wnt1-Cre-lineage marked cells. Together, these data support a model where Twist1 is required both for proper cNCC delamination, and for emigration from the dorsal neural tube and along cNCC migration pathways. Within the Twist1(-/-) neural crest cell populations that do emigrate to the OFT, a Hand-expressing subpopulation displays defective maturation, tracking, and, presumably, cell-cell adhesion, further compromising cNCC morphogenesis.