Embryonic stem (ES) cells possess the ability to renew themselves while 
maintaining the capacity to differentiate into virtually all cell types of the 
body. Current evidence suggests that ES cells maintain their pluripotent state 
by expressing a battery of transcription factors including Oct4 and Nanog. 
However, little is known about how ES cells maintain the expression of these 
pluripotent factors in ES cells. Here we present evidence that Oct4, Nanog, and 
FoxD3 form a negative feedback loop to maintain their expression in pluripotent 
ES cells. First, Oct4 maintains Nanog activity by directly activating its 
promoter at sub-steady-state concentration but repressing it at or above 
steady-state levels. On the other hand, FoxD3 behaves as a positive activator of 
Nanog to counter the repressive effect of Oct4. The expression of Oct4 is 
activated by FoxD3 and Nanog but repressed by Oct4 itself, thus, exerting an 
important negative feedback loop to limit its own activity. Indeed, 
overexpression of either FoxD3 or Nanog in ES cells failed to increase the 
concentration of Oct4 beyond the steady-state concentration, whereas knocking 
down either FoxD3 or Nanog reduces the expression of Oct4 in ES cells. Finally, 
overexpression of Oct4 or Nanog failed to compensate the loss of Nanog or Oct4, 
respectively, suggesting that both are required for ES self-renewal and 
pluripotency. Our results suggest the FoxD3-Nanog-Oct4 loop anchors an 
interdependent network of transcription factors that regulate stem cell 
pluripotency.