Müller glia (MG) are the only glial cell type produced by the neuroepithelial progenitor cells that generate the vertebrate retina. MG are required to maintain retinal homeostasis and support the survival of retinal neurons. Furthermore, in certain vertebrate classes, MG function as adult stem cells, mediating retinal regeneration in response to injury. However, the mechanisms that regulate MG development are poorly understood because there is considerable overlap in gene expression between retinal progenitor cells and differentiated MG. We show that the LIM homeodomain transcription factorLhx2is required for the development of MG in the mouse retina. Temporally controlled knock-out studies reveal a requirement forLhx2during all stages of MG development, ranging from the proliferation of gliocompetent retinal progenitors, activation of Müller-specific gene expression, and terminal differentiation of MG morphological features. We show that Lhx2 regulates gliogenesis in part by regulating directly the expression of Notch pathway genes includingNotch1,Dll1, andDll3and gliogenic transcription factors such asHes1,Hes5,Sox8, andRax. Conditional knock-out ofLhx2resulted in a rapid downregulation of Notch pathway genes and loss of Notch signaling. We further demonstrate that Müller gliogenesis induced by misexpression of the potently gliogenic Notch pathway transcriptional effectorHes5requiresLhx2expression. These results indicate that Lhx2 not only directly regulates expression of Notch signaling pathway components, but also acts together with the gliogenic Notch pathway to drive MG specification and differentiation.SIGNIFICANCE STATEMENTMüller glia (MG) are radial glial cells located in the vertebrate retina that are essential for the function and survival of retinal neurons. We found the LIM homeodomain transcription factor Lhx2 to be expressed in both retinal progenitor cells and MG. Using conditional knock-outs, we show thatLhx2is required during all stages of MG development. We also show that Lhx2 regulates directly the expression of components of the Notch signaling pathway, which promotes retinal Müller gliogenesis, as well as multiple gliogenic transcription factors. We further demonstrate thatLhx2is required for Hes5-dependent gliogenesis. This study identifiesLhx2as a central transcriptional regulator of both Notch-dependent and Notch-independent components of retinal gliogenesis.