Abstract Medulloblastoma (MB) is the most common primary childhood brain tumor. MB development frequently results from the dysregulation of sonic hedgehog and wingless pathways. In contrast to our understanding of these pathways, the pathogenic roles of aberrant epigenetic modifiers in MB remain little known. MLL4 is one of the most commonly mutated genes in MB. In our recent study, we showed that brain-specific knockout of the gene encoding the histone H3 lysine 4 methyltransferase MLL4 (also called COMPASS-like methyltransferase, KMT2D, MLL2, and ALR) induced spontaneous MBs. Mll4 loss highly upregulated several oncogenic signaling programs, including expression of several oncogenic Ras activators (e.g., Rasgrp1, Rasgrf1, and Rasgef1a). In contrast, Mll4 loss downregulated expression of tumor-suppressive genes, such as Dnmt3a. In this study, we sought to determine whether Mll4 loss cooperates with other oncogenic event in MB genesis. Particularly, PTCH (also known as PTCH1) is one of the most frequently mutated genes in MB, and its loss induces MB in mouse models. Thus, we examined the effect of Mll4 loss on of Ptch+/−-driven MB genesis. A heterozygous loss of Mll4 increased the incidence and progression of Ptch+/−-driven MB genesis. MBs were confirmed by H & E staining, and immunohistochemical staining using anti-Ki-67 showed that cell proliferation was markedly increased. Calbindin signals were undetectable in Nestin-Cre Mll4f/+ Ptch+/−, and NeuN levels were decreased in Nestin-Cre Mll4f/+ Ptch+/− cerebellum compared to Ptch+/− cerebellum, suggesting the impairment of Purkinje and granule cells. Mll4 loss reduced cerebellar granule cell differentiation. These findings indicate that a haploinsufficiency of MLL4 promotes Ptch+/−-driven MB genesis. This mouse model may be useful for future preclinical therapeutic experiments for the treatment of MB patients with MLL4 mutations. Citation Format: Shilpa S. Dhar, Dongyu Zhao, Tao Lin, Roy V. Sillitoe, Kaifu Chen, Min Gyu Lee. Heterozygous knockout of the histone modifier MLL4 promotes Ptch+/−-driven medulloblastoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1083.