Retinal stem cells existing within the adult mammalian ciliary epithelium offer tremendous potential for cell replacement therapies. However, these cells do not divide in vivo or spontaneously contribute to retinal regeneration following injury, and only following dissociation and subsequent culture does proliferation occur. Working from the hypothesis that adult retinal stem cell (aRSC) quiescence is induced by inhibitory signals secreted from one or more eye tissues, we cultured aRSCs either with or in conditioned media from tissue derived from various eye regions. We find that both co-culture and conditioned media from lens and cornea is highly effective in inhibiting aRSC sphere formation, with 25% conditioned media from either tissue reducing the number of primary spheres by at least 70%. In contrast conditioned media from ciliary epithelium, the in vivo niche for aRSCs, shows no effect on sphere formation. Interestingly, when we performed the same experiment using media from postnatal day 2 eyes, an age at which the retinal stem cell pool is still expanding, there was no effect on aRSC sphere formation. Taken together these findings suggest that signaling from specific regions of the adult eye act to inhibit proliferation of retinal stem cells present within the ciliary epithelium. In order to identify potential molecular pathways for this induction/maintenance of aRSC quiescence, we have taken a candidate molecule approach, initially focusing on Wnt, and BMP signaling. We find that inhibition of Wnt or stimulation of BMP signaling can both reduce the formation of primary spheres from aRSCs. However, these pathways have differential effects on the stem cell and progenitor cell populations. Specifically BMP manipulations appear to affect the proliferation of both stem cells and retinal progenitors, while Wnt signaling primarily affects stem cells. At present, we are combining both conditioned media and extrinsic signaling manipulation in order to identify the in vivo signals leading to aRSC quiescence.