To elucidate the epigenetic role of RNAi in mammals, we disrupted the gene for Eif2c2 (Ago2), which works as the sole slicer of RNAi in the Argonaute family. In mice, disruption of Eif2c2 leads to embryonic lethality early in development after the implantation stage. This phenotype is completely different from that in a previous report, but somewhat similar to the disruption of Dicer1, another important component of RNAi. We also show that Eif2c2 is not required for the maintenance of DNA methylation in imprinted genes, centromeric repeats, and Xist. This suggests that developmental defects in the Eif2c2-deficient mouse are caused not at the transcriptional level, but rather at the posttranscriptional level through the miRNA–protein complex.