The RFX transcription factor family target genes through conserved X-box sites and regulate gene networks associated with cilia formation. Within the brain, cilia regulate critical signalling pathways and are essential for brain patterning, neurogenesis, and neural migration. Recently, RFX3 has been implicated as a candidate gene for autism and related neurodevelopmental disorders (NDDs) following the identification of damaging, rare de novo variants in affected individuals. Although critical for normal brain development, the role of cilia and ciliogenesis in neurodevelopmental disorders remains unexplored. We therefore aim to determine the effects of RFX3 mutations on neurodevelopment in human models and explore the potential role of RFX target genes in the etiology of ASD through analysis of X-box variants. Using large-scale ASD cohort genome data comprising more than 5,800 families, we examined regulatory variants in X-box promotor motifs. We found that rare de novo X-box variants occurred at a rate ~2.5 fold higher in individuals with ASD compared to unaffected siblings. Additionally, luciferase reporter assays found that ASD-associated X-box promotor variants can significantly decrease gene expression. Utilising CRISPR/Cas9 edited hiPSCs we examined heterozygous (RFX3+/-) and homozygous (RFX3-/-) loss-of-function in 2D neural progenitor cultures and 3D cortical organoids. We found that RFX3-/- leads to reduced cilium length and both RFX3-/- and RFX3+/- conditions decreased activation of the ciliary signalling pathway, Sonic hedgehog, in neural progenitors. From single cell RNA-seq analysis of cortical organoids matured for 45 days, we identified a shift in cell type proportions with fewer early progenitor cells and more subcortical neurons found in RFX3-/- and RFX3+/- organoids. This preliminary analysis indicates that RFX3 loss-of-function leads to enhanced neurogenesis in cortical organoids, findings that we plan to validate using immunohistochemistry. Collectively, these data suggest that disruption of gene networks regulating ciliogenesis may contribute to the development and pathogenesis of autism and NDDs.