Mutations in the zinc finger transcription factor ZIC3 are associated with human left-right patterning abnormalities (X-linked heterotaxy, HTX1, MIM 306955), and mice null for Zic3 show a similar phenotype. However, the developmental function of Zic3 is largely unknown and its expression in early embryonic development suggests a role prior to organ formation. The current study of Zic3 null mice identifies a novel function for Zic3 in the gastrula-stage embryo. Analysis of Zic3 function at early embryonic stages shows that it ensures the fidelity of embryonic patterning, including patterning of the anterior visceral endoderm, the initiation of gastrulation, and positioning of the primitive streak. At later stages, deficiency of Zic3 results in abnormal mesoderm allocation. These results indicate a requirement for Zic3 during early embryogenesis prior to cardiac and visceral organ patterning.