Although Müller glial cells play pivotal roles in the vertebrate retina, the regulation of their development is poorly understood. While Notch-Hes5 signaling has been shown to be involved in this developmental process, the presence of Müller glial cells in Hes5-deficient mice suggests the involvement of other molecules. We found that two group E Sox genes, Sox8 and Sox9, are expressed in proliferating progenitors and then exclusively in Müller glial cells in mouse retina. Knocking-down Sox8 and Sox9 by shRNA significantly reduced the population of Müller glial cells and relatively increased the proportion of rod photoreceptors, suggesting that the Sox genes play roles in the specification of Müller glial cells. Using an activated form of Notch and the gamma-secretase inhibitor DAPT, we also found that Notch signaling regulates the transcription of Sox8 and Sox9. This is the first evidence that group E Sox genes play important roles in the developing vertebrate retina.