The cerebellum, traditionally associated with motor control, is now implicated also in higher cognitive functions. Patients exhibiting impaired cognition display cerebellar abnormalities, reported in disorders such as autism spectrum disorder (ASD), intellectual disability (ID), attention deficit hyperactivity disorder (ADHD), or schizophrenia. Purkinje cell migration deficits, volumetric changes and structural anomalies in the cerebellum have been observed in these conditions. This project aims to elucidate the connection between cerebellar circuit formation and neurodevelopmental disorders through the development of a novel tool for functional gene analysis. Using in ovo RNAi in E3 chicken embryos enables manipulation of candidate gene expression during development in a cell type-specific and temporally controlled manner. Here, we demonstrate that this technique is not limited to studies of commissural axon guidance in the developing spinal cord, or to the analysis of the peripheral nervous system, but can also be used to study circuit formation in the cerebellum. For validation of this technique, we replicated distinct phenotypes reported in prior studies, where knockdown of Endoglycan (Baeriswyl et al., 2022) or Axonin1/Contactin2 (Baeriswyl & Stoeckli, 2008) was achieved using ex ovo RNAi. Compared to ex ovo RNAi, our in ovo RNAi approach for manipulation of gene expression during neural circuit formation is higher throughput and more efficient with respect to embryonic survival.