Transgene and RNAi were used to establish five different FGF2-expressing monkey ear skin fibroblast (MESF) cell lines, the FGF2 over-expressed line (f1), the negative control of f1 (f2), RNA interfered line (f3), the negative control of f3 (f4) and the non-treated control (f5). The expression ratio of FGF2 in these lines was f1: f2: f3: f4: f5=4∶2∶1∶2∶2. The results indicated that c-fos, TGF-β1, INHBA, Gremlin1 were upregulated in f1 but downregulated in f3, while BMP4, TGF-β2 were downregulated in f1 but upregulated in f3, which implied that endogenous FGF2 affected the TGF-β signaling pathway and the expression level of related genes changed in MESFs. Further analysis of rhesus monkey embryonic stem cells (RhESCs) supported by these MESFs showed that RhESCs on f1 grew more quickly than those in other groups, in which the expression level of c-fos，TGF-β1，INHBA，Gremlin1，OCT-4，Nanog and Sox2 was higher, while that of BMP4 was lower; in contrast, RhESCs on f3 grew more slowly than in other groups, in which the expression level of c-fos，TGF-β1，INHBA，Gremlin1，OCT-4，Nanog and Sox2 declined, while that of BMP4 and TGF-β2 increased. EBs from these RhESCs expressed early markers representing all germ layers, but the expression levels of markers in RhESCs on f1 were lower than in other groups. These findings demonstrated that different FGF2 expression in feeder layers can not only influence expression of relative genes in MESFs, but also affect proliferation and self-renewal of ESCs.