Tid1 is the mammalian counterpart of the Drosophila tumor suppressor Tid56 and is also a DnaJ protein containing a conserved J domain through which it interacts with the heat shock protein 70 (Hsp70) family of chaperone proteins. We generated a Tid1 conditional mutation in mice, and the subsequent global removal of the Tid1 protein was achieved by crossing these conditional knockout mice with general deletor mice. No Tid1(-/-) embryos were detected as early as embryonic day 7.5 (E7.5). Nonetheless, Tid1-deficient blastocysts were viable, hatched, formed an inner cell mass and trophectoderm, and implanted (E4.5), suggesting that the homozygous mutant embryos die between E4.5 and E7.5. To assess the function of Tid1 in embryonic cells, mouse embryonic fibroblasts with the homologous Tid1 floxed allele were produced. Tid1 removal in these cells led to massive cell death. The death of Tid1-deficient cells could be rescued by ectopic expression of wild-type Tid1 but not by expression of the Tid1 protein that had a mutated J domain and was thus incapable of binding to Hsp70. We propose that Tid1 is critical for early mammalian development, most likely for its function in sustaining embryonic-cell survival, which requires its association with Hsp70.