Abstract: Comparative single-cell studies of group 3/4 medulloblastomas (MBs) and fetal brain have identified a common hierarchy of glutamatergic-lineage cell types and an apparent cell-of-origin in the rhombic lip. An increased understanding of the genetic regulators of stem-cell maintenance and differentiation in MB would clearly be of therapeutic benefit. There is a significant need for lineage-tracing systems to model MB malignant transformation, progression, and response to therapy. We developed in vivo and in organoid genetic lineage-tracing systems with a single-cell readout. A high-complexity lentiviral library expressing heritable polyadenylated molecular barcodes was transduced into D283 and D425 patient-derived cell lines. Barcoded cells were injected into the brains of immunocompromised mice. Both preimplantation barcoded cultures and the resulting mature tumors were profiled by single-cell RNA-sequencing (scRNA-seq). This captured both endogenous RNA and barcode transcripts. While only minimal barcode clash was detected in the preimplantation cultures, we observed a clonal expansion of barcodes in mature tumors which aligned with phylogenetics analysis of expressed mutations in endogenous RNA. Methods for assessing lineage coupling between transcriptional clusters and inferring their lineage relationship were developed. Ongoing efforts to recover barcodes from tumor sections via spatial transcriptomics will be presented. These studies fill a gap in status quo models of MB which are needed to leverage recent findings derived from single-cell analysis of human tumors.