Early mouse development is a complicated process that is controlled by various proteins including transcription factors. Recently, we identified a putative transcription factor, ELYS (embryonic large molecule derived from yolk sac), using a subtraction strategy. To elucidate the role of ELYS in vivo, we generated ELYS-deficient mice by homologous recombination. Although heterozygous mice appeared to be healthy, fertile and normal, embryos homozygous for the ELYS mutation died between embryonic day (E) 3.5 and 5.5. Null mutant blastocysts collected from the uterus at E3.5 were viable and indistinguishable from wild-type littermates. However, when cultured in vitro, they showed impaired proliferation of the inner cells, because of apoptosis. The expression of ELYS mRNA was detected in both the inner cell mass (ICM) and the trophectoderm at the blastocyst stage, and persisted throughout the developing embryo during E4.5 to 6.5. These results indicate that ELYS is a critical factor for early mouse development and is essential for the survival of the inner cells.