This study aimed to evaluate the pattern of gene expression induced by activin A in mouse Embryonic Stem Cells (ESCs). Mouse ES cells cultured in undifferentiated state by leukemia inhibitory factor and feeder layer cells. Following removing these two anti differentiation factors for 5 days and forming Embryoid Bodies (EBs), the cells divided to 8 equal cells per groups. Differentiation procedure was performed in a two staged protocol; Formed EBs for 4 days (Stage one); expanded differentiated ESCs on gelatin coated dishes for one week (stage two). In the stage one, the media of groups 2-7 contained 10, 30 and 100 ng mL(-1) Activin A. The media in stage two was the same for all groups and contained only Fetal Bovine Serum (FBS). The expression of undifferentiated, ectoderm, mesoderm and endoderm markers were compared with relative RT-PCR method and statistically analyzed. The expression of an undifferentiating marker; Nanog was increased in the Activin A treated groups of stage one. The expression of OCT4 reduced in Activin A treated groups in stage two. In the stage one, the expression of Nodal increased by Activin A. expression of sonic hedgehog (Shh) was suppressed in Activin A treated groups of both stages. In stage two, there were significant decrease for the expression of mesoderm (Brachyury) and Nodal and visceral endoderm (GATA4) markers (p < 0.01). The expression of definitive endoderm markers (PDX1, TAT) showed significantly increased in Activin A treated groups (p < 0.01). Activin A induced differentiation in high concentration by imbalance in undifferentiating markers. Nodal has a dual role, undifferentiating effect and regulation of visceral endoderm towards definitive endoderm. Overexpression of Nanog, alteration in the expression of Nodal and Shh inhibition are three mechanisms for explanation of differentiation induced by activin A in ES cells. These mechanisms induces cascade of gene expression that commits ESCs towards definitive endodermal cells.