Expression of differentiation marker genes is delayed in Klf6^-/- ES cells. (A) Differentiation into EBs of Klf6^+/+ ES cells, Klf6^+/- ES cells, and 2 clones of Klf6^-/- ES cells was analyzed. EBs differentiated for 2, 3, 4, 6, or 8 days were harvested at each time point. Total RNAs were extracted and analyzed by RT-PCR. The expression of the indicated genes is shown. The cDNAs generated from the same mRNAs were also subjected to quantitative real-time PCR for (B) Klf6, (C) Rex1, (D) Brachyury, (E) Scl, (F) Klf1, (G) Gata1, and (H) Gata2.