Cyclin-dependent kinase-like 5 (CDKL5) is an X-linked gene associated with early infantile epileptic encephalopathy, atypical Rett Syndrome, and autism spectrum disorders. Patients with CDKL5 mutations display a heterogeneous array of clinical symptoms, the most prominent of which include early-onset epileptic seizures, intellectual disability, marked hypotonia, and autistic features. Despite the strong genetic linkage between CDKL5 mutations and neurodevelopmental disorders, the biological function of CDKL5 and its pathogenic mechanisms remain largely uncharacterized. Consequently, treatments for CDKL5 disorder have been largely ineffective and limited to symptom management. This study aims to dissect the molecular and cellular basis of CDKL5 disorder using novel mouse models and to identify signaling pathways that can be targeted for therapeutic development. To determine the genetic causality of CDKL5 disorder, we generated the first Cdkl5 knockout (KO) mouse and found that mice lacking CDKL5 mimic key symptoms of CDKL5 disorder, including impaired motor control, poor learning and memory, and autistic-like behaviors. KO mice also show deficits in neural circuit communication and alterations in many signal transduction pathways, but do not develop spontaneous seizures. Previous studies using in vitro cell cultures and shRNA-mediated knockdown have implicated CDKL5 in dendritic morphogenesis and the phosphorylation of MeCP2, mutations of which cause Rett Syndrome, but the results of these studies are contentious. Therefore, to understand the in vivo function of CDKL5 and to investigate how loss of CDKL5 contributes to epileptic and autistic features, we have generated Cdkl5 conditional KO mice. Notably, our studies show that selective deletion of Cdkl5 from forebrain GABAergic neurons (Dlx-cKO) recapitulates autistic-like phenotypes, whereas selective removal of Cdkl5 from forebrain glutamatergic neurons (Nex-cKO) results in the development of spontaneous seizures. The separation of distinct aspects of CDKL5 disorder-related phenotypes in the two complementary conditional KO lines suggests that the epileptic and autistic phenotypes of CDKL5 disorder are mediated by distinct neural circuits and that loss of CDKL5 in specific cell types may differentially disrupt signaling pathways and impair neuronal function.