A novel paired-like homeobox gene, designated Esx1, was isolated in a screen for homeobox genes that regulate mouse embryogenesis. Analysis of a mouse interspecific backcross panel demonstrated that Esx1 mapped to the distal arm of the X chromosome. During embryogenesis, Esx1 expression was restricted to extraembryonic tissues, including the endoderm of the visceral yolk sac, the ectoderm of the chorion, and subsequently the labyrinthine trophoblast of the chorioallantoic placenta. In adult tissues, Esx1 expression was detected only in testes. However, Esx1 transcripts were not detected in the testes of sterile W/Wv mice, suggesting that Esx1 expression is restricted to male germ cells. In situ hybridization experiments of testes indicated that Esx1 transcripts were most abundant in pre- and postmeiotic germ cells. Hybridization experiments suggested that Esx1 was conserved among vertebrates, including amphibians, birds, and mammals. During mouse development, the paternally derived X chromosome is preferentially inactivated in extraembryonic tissues of XX embryos, including the trophoblast, visceral endoderm, and parietal endoderm. In addition, the X chromosome is transiently inactivated during the meiotic stages of spermatogenesis. Thus, the identification of Esx1 provides a molecular entry point into a genetic pathway to understand X chromosome-regulated fetal-maternal interactions and male germ cell development.