The study investigates the role of Activin/Nodal and FGF pathways in maintaining the pluripotency of human embryonic stem cells (hESCs). The authors found that Activin/Nodal signaling through Smad2/3 activation is essential for maintaining hESC pluripotency. Inhibition of Activin/Nodal signaling using follistatin, Lefty, Cerberus-Short, or 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. The FGF signaling pathway, which maintains long-term expression of pluripotency markers, also plays a crucial role. Inhibition of FGF signaling with the tyrosine kinase inhibitor SU5402 causes hESC differentiation, but this effect depends on Activin/Nodal signaling. Combining Activin or Nodal with FGF2 in a chemically defined medium without serum, feeder cells, or Serum Replacer can maintain long-term in vitro pluripotency. These findings suggest that the Activin/Nodal pathway maintains pluripotency through mechanisms involving FGF as a competence factor, providing evidence for distinct mechanisms of pluripotency maintenance in mouse and human ESCs.The study investigates the role of Activin/Nodal and FGF pathways in maintaining the pluripotency of human embryonic stem cells (hESCs). The authors found that Activin/Nodal signaling through Smad2/3 activation is essential for maintaining hESC pluripotency. Inhibition of Activin/Nodal signaling using follistatin, Lefty, Cerberus-Short, or 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. The FGF signaling pathway, which maintains long-term expression of pluripotency markers, also plays a crucial role. Inhibition of FGF signaling with the tyrosine kinase inhibitor SU5402 causes hESC differentiation, but this effect depends on Activin/Nodal signaling. Combining Activin or Nodal with FGF2 in a chemically defined medium without serum, feeder cells, or Serum Replacer can maintain long-term in vitro pluripotency. These findings suggest that the Activin/Nodal pathway maintains pluripotency through mechanisms involving FGF as a competence factor, providing evidence for distinct mechanisms of pluripotency maintenance in mouse and human ESCs.