Abstract Oligodendrogenesis is a complex process that requires changes in transcription factor expression for the generation, expansion, and differentiation of oligodendrocyte progenitor cells (OPCs) into mature oligodendrocytes (OLs) that produce myelin. The overall goal of this proposal is to focus on understanding the diversity of the OPC stage in the process. Our gene expression screen to identity novel OPC markers revealed the homeobox gene Gsx1 exhibits temporal expression in OPCs during development. Here we propose to develop new mouse models to address the role of Gsx1 in OPC biology. In the first aim, we will test if Gsx1 is a stage specific regulator in OPCs and generate an OPC specific conditional mutant to determine if altering Gsx1 gene function causes changes in OPC expansion, OL differentiation, and/or myelination. We will also test if the closely related gene, Gsx2, compensates for the loss of Gsx1 in OPCs. In the second aim, we will test if sustained Gsx1 expression throughout OL development promotes OPC generation and subsequently OL differentiation and myelination using a temporally controlled doxycycline regulated gain-of-function model. In the third aim, we will utilize the Gsx1 lineage to reveal OPC diversity during postnatal oligodendrogenesis by generating a novel Gsx1 reporter allele using Timer Fluorescent Protein. These studies will identify the specific roles of Gsx1 during the OPC stage of OL development and provide new mouse models key for understanding the complex regulation of OPCs during oligodendrogenesis.