It is now widely accepted that tumors are composed of heterogeneous population of cells, which contribute to many aspects of treatment resistance observed in clinic. Despite the acknowledgment of the tumor cell heterogeneity, little evidence was shown about complexity and dynamics of this heterogeneity in vivo, mainly because of lacking flexible genetic tools which allow sophisticated analysis in primary tumors. We recently developed a very efficient mouse somatic brain tumor model which have a full penetrance of high grade glioma development. Combination of this model with several transgenic mouse lines allow us to isolate and track different population of cells in primary tumors, most importantly, we also confirmed that this can be done on single cell level. Here I propose to use this set of valuable genetic tools to dissect the cellular heterogeneity in mouse gliomas. First we will perform several single cell lineage tracing experiment to demonstrate the contribution of brain tumor stem cell, tumor progenitors as well as the relatively differentiated cells, which will provide a complete data sets of clonal dynamics of different tumor cell types.Second we will further perform this tracing experiment with the presence of conventional chemotherapy.Third we will perform single cell RNA sequencing experiment to capture the molecular signature, which determines the cellular heterogeneity, discovered by single cell tracing. This result will be further validated by analysis of this molecular signatures in human primary tumors. We will also use our established in vivo target validation approach to manipulate the candidate molecular regulators to establish the functional correlation between molecular signature and phenotypic heterogeneity. This project will greatly improve our understanding of tumor heterogeneity, and possibly provide novel approaches and strategies of targeting human glioblastomas.