Neurodevelopmental disorders (NDDs) affect brain function and development, with 90% lacking approved treatments. Understanding their pathogenic mechanisms is critical for developing precision gene therapies. An autosomal recessive NDD associated with variants in the Mediator complex subunit 27 (MED27) gene is previously identified. The Mediator complex is essential for transcription initiation by bridging transcription factors (TFs) at enhancers to RNA polymerase II at promoters. All patients with MED27 variants exhibit cerebellar hypoplasia or atrophy, underscoring the cerebellum's heightened vulnerability to MED27 dysfunction. To investigate the disease mechanisms, in vitro stem cells carrying patient-specific MED27 variants and in vivo mouse models with Med27 loss-of-function (LoF) are generated. These preclinical models recapitulate key patient phenotypes, including progressive cerebellar atrophy and motor deficits. Molecular analyses reveal that mutant MED27 destabilizes the Mediator complex, impairing its chromatin occupancy and altering chromatin interactions. Comprehensive transcriptomic profiling, including single-cell resolution spatial transcriptomics, identifies dysregulation of downstream targets regulated by MED27, such as critical master regulatory TFs involved in neurogenesis and cerebellar development. This study elucidates a partial LoF mechanism underlying MED27-associated NDDs and establishes a prototype for investigating NDDs caused by pathogenic variants in Mediator subunits.