How do the evolutionarily young genes facilitate the generation of new traits is one of the fundamental scientific questions in evolutionary and developmental biology.The processes of the differentiations from Embryonic Stem Cell (ESC) to various types of terminal-differentiated somatic cells are the basis of embryonic development.Understanding of the roles of young genes in the ESC differentiation is of great significance.In this study,based on the published epigenomic,transcriptomic and proteomic datas of human and mouse ESC differentiation,we firstly explored the expression pattern of young genes during the differentiation of ESC into three germ layers.Interestingly,among the stage-specific genes in each lineage,the distribution pattern of young genes is different.Its obvious that young genes tend to be expressed in the early stage of the differentiation into ectoderm.On the contrary,young genes tend to be expressed in the late stage of the differentiation into endoderm.Secondly,the key young-protein-containing modules and pathways regulating differentiation of ESC were explored by k-mean clustering and weighted correlation network analysis (WGCNA) methods.The functions of these modules and pathways are related to the evolutionarily novel characteristics.Our results provide a global understanding of the roles of young genes in the ESC differentiation,and it is important to the further understanding of the evolution of developmental mechanisms.