Fig. 5. Primitive endoderm specification of mouse ES cells can be induced solely by cellular aggregation regardless of the presence of external factors (LIF and BMP4), which are sufficient to maintain self-renewal of ES cells in monolayer culture (top). ES-cell aggregation induces repression of the Nanog gene in the outer layer independent of the LIF/STAT3 and BMP4/Smads pathways (Cavaleri and Schöler, 2003; Ying et al., 2003). The repression of Nanog subsequently leads to ES cells differentiating into the primitive endoderm (bottom).