Proteins containing "cold shock" domains belong to the most evolutionarily conserved family of nucleic acid-binding proteins known among bacteria, plants, and animals. One of these proteins, YB-1, is widely expressed throughout development and has been implicated as a cell survival factor that regulates the transcription and/or translation of many cellular growth and death-related genes. For these reasons, YB-1 deficiency has been predicted to be incompatible with cell survival. However, the majority of YB-1-/- embryos develop normally up to embryonic day 13.5 (E13.5). After E13.5, YB-1-/- embryos exhibit severe growth retardation and progressive mortality, revealing a nonredundant role of YB-1 in late embryonic development. Fibroblasts derived from YB-1-/- embryos displayed a normal rate of protein synthesis and minimal alterations in the transcriptome and proteome but demonstrated reduced abilities to respond to oxidative, genotoxic, and oncogene-induced stresses. YB-1-/- cells under oxidative stress expressed high levels of the G1-specific CDK inhibitors p16Ink4a and p21Cip1 and senesced prematurely; this defect was corrected by knocking down CDK inhibitor levels with specific small interfering RNAs. These data suggest that YB-1 normally represses the transcription of CDK inhibitors, making it an important component of the cellular stress response signaling pathway.