TP53 is the most frequently mutated tumor suppressor gene in human cancer, with nearly 50% of all tumors exhibiting a loss-of-function mutation. To further elucidate the genetic pathways involving TP53 and cancer, we have exploited the zebrafish, a powerful vertebrate model system that is amenable to whole-genome forward-genetic analysis and synthetic-lethal screens. Zebrafish lines harboring missense mutations in the tp53 DNA-binding domain were identified by using a target-selected mutagenesis strategy. Homozygous mutant fish from two of these lines were viable and exhibited mutations similar to those found in human cancers (tp53^N168K and tp53^M214K). Although homozygous tp53^N168K mutants were temperature-sensitive and suppressed radiation-induced apoptosis only at 37°C, cells in the tp53^M214K embryos failed to undergo apoptosis in response to γ radiation at both 28 and 37°C. Unlike wild-type control embryos, irradiated tp53^M214K embryos also failed to up-regulate p21 and did not arrest at the G₁/S checkpoint. Beginning at 8.5 months of age, 28% of tp53^M214K mutant fish developed malignant peripheral nerve sheath tumors. In addition to providing a model for studying the molecular pathogenic pathways of malignant peripheral nerve sheath tumors, these mutant zebrafish lines provide a unique platform for modifier screens to identify genetic mutations or small molecules that affect tp53-related pathways, including apoptosis, cell-cycle delay, and tumor suppression.