Both inhibitors of JNK and ERK retarded ES differentiation. After treatment with 10 μM SP600125, 2 or 10 μM U0126 from days 1-3, ES cells were plated on 0.1% matrigel-coated glasses and stained with anti-Oct4 (A) and anti-Nanog antibodies (B) on day 6. The ratio of undifferentiated pluripotent ES cells to total DAPI+ cells (n>500 cells) was estimated from experiments done in triplicate. Brachyury (T) (C), Sox17 (D) and cytokeratin 14 (E) expressions, representing mesodermal, endodermal and surface ectodermal cell lineages respectively, were examined in ES cells on day 6 with SFEB treatment. Nuclei of all cells are seen by DAPI staining in blue. The statistic results of the cell numbers in panel C and D were also estimated, respectively (E, F).