The role of individual ocular tissues in mediating changes to the sclera during myopia development is unclear. The aim of this study was to examine the effects of retina, RPE and choroidal tissues from myopic and hyperopic chick eyes on the DNA and glycosaminoglycan (GAG) content in cultures of chick scleral fibroblasts. Primary cultures of fibroblastic cells expressing vimentin and α-smooth muscle actin were established in serum-supplemented growth medium from 8-day-old normal chick sclera. The fibroblasts were subsequently co-cultured with posterior eye cup tissue (full thickness containing retina, RPE and choroid) obtained from untreated eyes and eyes wearing translucent diffusers (form-deprivation myopia, FDM) or -15D lenses (lens-induced myopia, LIM) for 3 days (post-hatch day 5-8) (n = 6 per treatment group). The effect of tissues (full thickness and individual retina, RPE, and choroid layers) from -15D (LIM) versus +15D (lens-induced hyperopia, LIH) treated eyes was also determined. Refraction changes in the direction predicted by the visual treatments were confirmed by retinoscopy prior to tissue collection. Glycosaminoglycan (GAG) and DNA content of the scleral fibroblast cultures were measured using GAG and PicoGreen assays. There was no significant difference in the effect of full thickness tissue from either FDM or LIM treated eyes on DNA and GAG content of scleral fibroblasts (DNA 8.9 ± 2.6 μg and 8.4 ± 1.1 μg, p = 0.12; GAG 11.2 ± 0.6 μg and 10.1 ± 1.0 μg, p = 0.34). Retina from LIM eyes did not alter fibroblast DNA or GAG content compared to retina from LIH eyes (DNA 27.2 ± 1.7 μg versus 23.2 ± 1.5 μg, p = 0.21; GAG 28.1 ± 1.7 μg versus. 28.7 ± 1.2 μg, p = 0.46). Similarly, the choroid from LIH and LIM eyes did not produce a differential effect on DNA content (DNA LIM 46.9 ± 6.4 versus LIH 51.5 ± 4.7 μg, p = 0.31). In contrast, scleral fibroblast DNA was greater in co-culture with RPE from LIM eyes than the empty basket and DNA content less for co-culture with RPE from LIH eyes (LIM: 72.4 ± 6.3 μg versus empty basket: 46.03 ± 1.0 μg; p = 0.0005 and LIH: 27.9 ± 2.3 μg versus empty basket: 46.03 ± 1.0 μg; p = 0.0004). GAG content was lower with RPE from LIM eyes (LIM: 27.7 ± 0.9 μg versus empty basket: 29.5 ± 0.8 μg, p = 0.021) and was higher with RPE from LIH eyes (LIH: 33.7 ± 1.9 μg versus empty basket: 29.5 ± 0.8 μg, p = 0.010). Choroid from LIM eyes induce a relative increase in scleral GAG content e.g. (LIM: 32.5 ± 0.7 μg versus empty basket: 29.5 ± 0.8 μg, p = 0.0004) while, choroid from LIH eyes induced a relative decrease in scleral GAG content (LIH: 18.9 ± 1.2 μg versus empty basket: 29.5 ± 0.8 μg, p = 0.0034). GAG content of cells in co-culture with choroid from LIM versus LIH treated eyes was significantly different (32.5 ± 0.7 μg versus 18.9 ± 1.2 μg respectively, p = 0.0002). In conclusion, these experiments provide an evidence for a directional growth signal that is present (and remains) in the ex-vivo RPE/choroid, but that does not remain in the ex-vivo retina. The identity of this factor(s) that can modify scleral cell DNA and GAG content requires further research.