The study shows that the Activin/Nodal and FGF pathways work together to maintain the pluripotency of human embryonic stem cells (hESCs). Activin/Nodal signaling through Smad2/3 activation is essential for maintaining the pluripotent status of hESCs. Inhibition of Activin/Nodal signaling by follistatin, Lefty, or Cerberus-Short, or by the Activin receptor inhibitor SB431542, leads to hESC differentiation. However, neither Nodal nor Activin alone is sufficient to sustain long-term hESC growth in a chemically defined medium without serum. FGF2 can also maintain long-term expression of pluripotency markers, but its effect on hESC pluripotency depends on Activin/Nodal signaling. The combination of Activin or Nodal plus FGF2 in the absence of feeder-cell layers, conditioned medium, or serum can achieve long-term maintenance of in vitro pluripotency. These findings suggest that the Activin/Nodal pathway maintains pluripotency through mechanisms in which FGF acts as a competence factor, providing further evidence of distinct mechanisms for preserving pluripotency in mouse and human ESCs. The study also shows that FGF can maintain Cripto expression in hESCs, but cannot be replaced by recombinant Cripto. FGF does not appear to act by controlling the nuclear localisation of Phospho-Smad2 or Phospho-Smad1/5/8. The study provides substantial evidence that Activin/Nodal signaling plays a key role in maintaining pluripotency of hESCs, and that FGF signaling pathway also plays a role in maintaining pluripotency of hESCs. The findings suggest that the Activin/Nodal pathway is essential to maintain pluripotency of hESCs.The study shows that the Activin/Nodal and FGF pathways work together to maintain the pluripotency of human embryonic stem cells (hESCs). Activin/Nodal signaling through Smad2/3 activation is essential for maintaining the pluripotent status of hESCs. Inhibition of Activin/Nodal signaling by follistatin, Lefty, or Cerberus-Short, or by the Activin receptor inhibitor SB431542, leads to hESC differentiation. However, neither Nodal nor Activin alone is sufficient to sustain long-term hESC growth in a chemically defined medium without serum. FGF2 can also maintain long-term expression of pluripotency markers, but its effect on hESC pluripotency depends on Activin/Nodal signaling. The combination of Activin or Nodal plus FGF2 in the absence of feeder-cell layers, conditioned medium, or serum can achieve long-term maintenance of in vitro pluripotency. These findings suggest that the Activin/Nodal pathway maintains pluripotency through mechanisms in which FGF acts as a competence factor, providing further evidence of distinct mechanisms for preserving pluripotency in mouse and human ESCs. The study also shows that FGF can maintain Cripto expression in hESCs, but cannot be replaced by recombinant Cripto. FGF does not appear to act by controlling the nuclear localisation of Phospho-Smad2 or Phospho-Smad1/5/8. The study provides substantial evidence that Activin/Nodal signaling plays a key role in maintaining pluripotency of hESCs, and that FGF signaling pathway also plays a role in maintaining pluripotency of hESCs. The findings suggest that the Activin/Nodal pathway is essential to maintain pluripotency of hESCs.