Significance We have previously shown that novel APOBEC1 target 1 (NAT1), which is homologous to the C-terminal two thirds of eukaryotic translation initiation factor 4G (eIF4G), is essential for differentiation of mouse embryonic stem cells (mES cells). In the current study, we demonstrate that Nat1 supports the translation of mitogen-activated protein kinase kinase kinase 3 ( Map3k3 ) and son of sevenless homolog 1 ( Sos1 ). Deletion of Nat1 resulted in decreased protein levels of these factors, resulting in suppression of the Erk and Akt signaling pathway. Forced expression of Map3k3 induced differentiation in Nat1 -null mES cells. Thus Nat1 is involved in the translation of proteins that are required for the differentiation of mES cells.