Stem cells are unspecialized cells and excellent model in developmental biology and a
promising approach to the treatment of disease and injury. In the last 30 years, pluripotent embryonic
stem (ES) cells were established from murine and primate sources, and display indefinite replicative
potential and the ability to differentiate to all three embryonic germ layers. Despite large efforts in
many aspects of rodent and non-rodent pluripotent stem cell culture, a number of diverse challenges
remain. Natural and synthetic small molecules (SMs) strategy has the potential to overcome these hurdles.
Small molecules are typically fast and reversible that target specific signaling pathways, epigenetic
processes and other cellular processes. Inhibition of the transforming growth factor-β (TGF-β/Smad)
and fibroblast growth factor 4 (FGF4)/ERK signaling pathways by SB431542 and PD0325901 small
molecules, respectively, known as R2i, enhances the efficiency of mouse, rat, and chicken pluripotent
stem cells passaging from different genetic backgrounds. Therefore, the application of SM inhibitors of
TGF-β and ERK1/2 with leukemia inhibitory factor (LIF) allows the cultivation of pluripotent stem
cells in a chemically defined condition. In this review, we discuss recently emerging evidence that dual
inhibition of TGF-β and FGF signaling pathways plays an important role in regulating pluripotency in
both rodent and non-rodent pluripotent stem cells.