PURPOSE. Although some of the factors and signaling pathways that are involved in induction of fiber differentiation have been defined, such as FGF-mediated MAPK/ERK and PI3-K/Akt signaling, the factors in the vitreous that regulate this differentiation process in vivo have yet to be identified. The purpose of this study was to better understand the role of growth factors in vitreous that regulate this process by further characterizing the signaling pathways involved in lens fiber differentiation. METHODS. Rat lens epithelial explants were used to compare the ability of vitreous, IGF-1, PDGF-A, EGF, and FGF-2 to stimulate the phosphorylation of ERK1/2 and Akt leading to fiber differentiation, in the presence or absence of selective receptor tyrosine kinase (RTK) inhibitors. RESULTS. Similar to vitreous, FGF induced a sustained ERK1/2 signaling profile, unlike IGF, PDGF, and EGF, which induced a more transient (shorter) activation of ERK1/2. For Akt activation, IGF was the only factor that induced a profile similar to vitreous. IGF, PDGF, and EGF potentiated the effects of a low dose of FGF on lens fiber differentiation by extending the duration of ERK1/2 phosphorylation. In the presence of selective RTK inhibitors, although the sustained vitreous-induced ERK1/2 signaling profile and subsequent fiber differentiation was perturbed, the results also showed that, although prolonged ERK1/2 phosphorylation was necessary, it was not sufficient for fiber differentiation to proceed. CONCLUSIONS. These results are consistent with FGF's being the key growth factor involved in vitreous-induced signaling leading to lens fiber differentiation; however, they also indicate that other vitreal growth factors such as IGF may be involved in fine-tuning ERK1/2- and Akt-phosphorylation to the level that is necessary for initiation and/or maintenance of lens fiber differentiation in vivo.