In humans, low expression of DIAPH3 is associated with poor prognosis in glioblastoma patients. In mice, DIAPH3 loss induces aberrant mitosis, chromosomal instability, and cell death; whereas the combined loss of DIAPH3 and p53 causes high-grade diffuse glioma and accelerates tumor onset. In this study, we used Diaph3/p53 double conditional knockout mice (dcKO) and ultra-high-resolution MRI to detect and characterize the development and progression of glioma. We transcriptionally profiled early and advanced gliomas and identified stage-dependent alterations in molecular signatures as the tumor progresses. Notably, genetic pathways underlying self-renewal and extracellular matrix (ECM) interactions were upregulated at the expense of neural activity signature, which was severely shrunk. CKAP2L was identified as a differentially expressed gene at both early and advanced tumor stages in the dcKO mice. A subsequent siRNA screen to identify genes involved in proliferation and migration of glioblastoma cells identified CKAP2L as a top hit. Functional studies showed that CKAP2L depletion reduced proliferation and migration and induced cell accumulation in G₂/M phase of the cell cycle. We also used data mining and thoroughly characterized the relationship between CKAP2L expression and glioma development. We found that CKAP2L expression correlates with proliferative and progenitor-associated markers, pointing to a potential role in maintaining highly proliferative and progenitor-like tumor cell state. We also identified a differentially methylated CpG site in the CKAP2L promoter that was associated with changes in gene expression across glioma datasets. Overall, our findings show that CKAP2L may contribute to glioma progression, help distinguish tumor grade, and represent a potential therapeutic target in glioblastoma treatment.