Objective: Several mouse models of cardiac neural crest cell (NCC)-associated conotruncal heart defects exist, but the specific cellular and molecular defects within cardiac NCC morphogenesis remain largely unknown. Our objective was to investigate the underlying mechanisms resulting in outflow tract defects and why insufficient cardiac NCC reach the heart of the Splotch (Sp^2H) mouse mutant embryo. Methods: For this study we used in vitro cell culture techniques, in vivo mouse–chick chimeras, BrdU cell proliferation labeling, TUNEL labeling to visualize apoptosis and the molecular markers AP-2, Wnt-1 and Wnt-3a to characterize NCC morphogenesis in vivo. Results: Expression of the NCC marker AP-2 revealed an extensive reduction in migratory NCC, however the rates of cell proliferation and apoptosis were unaffected, and do not account for the Sp^2H NCC-associated heart defects. Further expression analysis revealed that Wnt-1, but not Wnt-3a, is expressed at decreased levels within Sp^2H and that the cardiac NCC fail to undergo normal NC stem cell proliferative expansion prior to migration while still in the neural folds. However, when placed into a wild-type matrix or a tissue culture environment, the Sp^2H cardiac NCC could migrate normally. Additionally, this reduced population of Sp^2H NC stem cells do migrate properly within the Sp^2H environment, as observed by neurofilament expression and cardiac innervation. Conclusions:. Taken together, all these data indicate that the Sp^2H defect is intrinsic to the NC stem cells themselves and that there is a decrease in the number of pre-migratory cardiac NCC that form. It appears that this decrease in NCC number is the primary defect that ultimately leads to a lack of a cardiac NCC-derived Sp^2H outflow tract septum.