Paediatric high grade gliomas (pHGGs) are uniformly fatal childhood central nervous system (CNS) tumours. More than 30% of pHGGs are classified as diffuse hemispheric glioma (DHG) H3.3 G34R/V mutant and arise in the cerebral hemispheres of adolescents and young adults. The median survival rate ranges from 15-22 months post treatment. DHG is molecularly defined by a heterozygous driver mutation in the H3F3A gene which encodes the histone variant H3.3, resulting in the substitution of a glycine for an arginine or valine at residue 34. H3.3 G34R/V driver mutations can co-occur with other mutations including TP53, ATRX and amplification of PDGFRA and MYCN. DHGs have recently been defined by transcriptional profiles and tumour cells recapitulate GABAergic interneuron precursor signatures. During human forebrain development, newborn GABAergic interneurons migrate tangentially from the ganglionic eminences of the ventral forebrain to populate the dorsal neocortex, thereby contributing to the balance of inhibitory and excitatory neurons. Further, the GABAergic progenitor-like cell of origin for DHG most accurately resembles the molecular niche of the ventral forebrain. Although current understanding of neurodevelopmental biology derives from seminal studies in mouse models, new technologies have emerged to study human brain development. We aim to model the DHG neurodevelopmental milieu with human pluripotent stem cell (hPSC) derived ventral forebrain organoids. We will express the epigenetic driver mutation of DHG, H3.3G34R in forebrain organoids to model the neurodevelopmental origins of this disease. We will then investigate the effect of the H3.3G34R mutation by analysing differences in transcription and chromatin accessibility to uncover the mechanisms involved in DHG formation. We have established protocols to grow ventral and dorsal forebrain organoids. We have employed NKX2.1-GFP reporter cell lines to track differentiation of ventral forebrain organoids with expression beginning at Day 14. Further, we have designed an imaging protocol to validate expression of other stem cell neural progenitor markers such as NESTIN, FOXG1, GABA and DLX2. By employing a human specific model of ventral forebrain development, this research aims to uncover the mechanisms involved in the formation of DHG.