Forkhead box L2 (FOXL2) is a member of the forkhead nuclear factor 3 gene family and plays an essential role in ovarian growth and maturation in mammals. However, its potential effects and regulative mechanism in development of chicken ovarian prehierarchical follicles remain unexplored. In this study, the cooperative effects of FOXL2 with activin A, growth differentiation factor-9 (GDF9) and follistatin, three members of the transforming growth factor beta (TGF-β) superfamily that were previously suggested to exert a critical role in follicle development was investigated. We demonstrated herein, using in-situ hybridization, Northern blot and immunohistochemical analyses of oocytes and granulosa cells in various sizes of prehierarchical follicles that both FOXL2 transcripts and FOXL2 proteins are predominantly expressed in a highly similar expression pattern to that of GDF9 gene. In addition, the FOXL2 transcript was found at lower levels in theca cells in the absence of GDF9. Furthermore, culture of granulosa cells (GCs) from the prehierarchical follicles (6-8 mm) in conditioned medium revealed that in the pcDNA3.0-FOXL2 transfected GCs, there was a more dramatic increase in FSHR mRNA expression after treatment with activin A (10 ng/ml) or GDF9 (100 ng/ml) for 24 h which caused a stimulatory effect on the GC proliferation. In contrast, a significant decrease of FSHR mRNA was detected after treatment with follistatin (50 ng/ml) and resulted in an inhibitory effect on the cell proliferation. The results of this suggested that FOXL2 plays a bidirectional modulating role involved in the intracellular FSHR transcription and GC proliferation via an autocrine regulatory mechanism in a positive or negative manner through cooperation with activin A and/or GDF9, and follistatin in the hen follicle development. This cooperative action may be mediated by the examined Smad signals and simultaneously implicated in modulation of the StAR, CCND2, and CYP11A1 expression.