SUMMARY Cancer cell-of-origin is difficult to identify by analyzing cells within terminal-stage tumors, whose identity could be concealed by the acquired plasticity. Thus an ideal approach to identify the cell-of-origin is to analyze proliferative abnormalities in distinct lineages prior to malignancy. Here we use Mosaic Analysis with Double Markers (MADM) in mice to model gliomagenesis by initiating concurrent p53/Nf1 mutations sporadically in neural stem cells (NSCs). Surprisingly, MADM-based lineage tracing revealed significant aberrant growth prior to malignancy only in oligodendrocyte precursor cells (OPCs), but not in any other NSC-derived lineages or NSCs themselves. Upon tumor formation, phenotypic and transcriptome analyses of tumor cells revealed salient OPC features. Finally, introducing the same p53/Nf1 mutations directly into OPCs consistently led to gliomagenesis. Our findings suggest OPCs as the cell-of-origin in this model even when initial mutations occur in NSCs, and highlight the importance of analyzing pre-malignant stages to identify the cancer cell-of-origin.