The ability to detect and degrade transcripts that lack full coding potential is ubiquitous but non-essential in lower eukaryotes, leaving in question the evolutionary basis for complete maintenance of this function. One hypothesis holds that nonsense-mediated RNA decay (NMD) protects the organism by preventing the translation of truncated peptides with dominant negative or deleterious gain-of-function potential. All organisms studied to date that are competent for NMD express a structural homolog of Saccharomyces cerevisiae Upf1p. We have now explored the consequences of loss of NMD function in vertebrates through targeted disruption of the Rent1 gene in murine embryonic stem cells which encodes a mammalian ortholog of Upf1p. Mice heterozygous for the targeted allele showed no apparent phenotypic abnormalities but homozygosity was never observed, demonstrating that Rent1 is essential for embryonic viability. Homozygous targeted embryos show complete loss of NMD and are viable in the pre-implantation period, but resorb shortly after implantation. Furthermore, Rent1(-/-) blastocysts isolated at 3.5 days post-coitum undergo apoptosis in culture following a brief phase of cellular expansion. These data suggest that NMD is essential for mammalian cellular viability and support a critical role for the pathway in the regulated expression of selected physiologic transcripts.