We analyzed the transcriptional profiles of differentiating mouse embryonic stem cells (mESCs) and show that embryoid bodies (EBs) sequentially expressed genes associated with the epiblast, primitive streak, mesoderm and endoderm of the developing embryo, validating ESCs as a model system for identifying cohorts of genes marking specific stages of embryogenesis. By comparing the transcriptional profiles of undifferentiated ESCs to those of their differentiated progeny, we identified 503 mESC and 983 hESC genes selectively expressed in undifferentiated ES cells. Over 75% of the mESC genes were expressed in hESC and vice versa, attesting to the underlying similarity of mESCs and hESCs. The expression of a cohort of 68 genes decreased greater than 2-fold during differentiation in both mESCs and hESCs. As well as containing many validated ESC genes such as Oct4 [Pou5f1], Nanog and Nodal, this cohort included an uncharacterised gene (FLJ30046), which we designated SLAIN1/Slain1. Slain1 was expressed at the stem cell and epiblast stages of ESC differentiation and in the epiblast, nervous system, tailbud and somites of the developing mouse embryo. SLAIN1 and its more widely expressed homologue SLAIN2 comprise a new family of structurally unique genes conserved throughout vertebrate evolution.