Summary Mutations in the X-linked gene, methyl-CpG binding protein 2 (MECP2), underlie a wide range of neuropsychiatric disorders, most commonly Rett syndrome (RTT), a severe neurodevelopmental disorder. Despite numerous studies, the question of why the loss of MeCP2 results in RTT remains largely unanswered, and it represents a major challenge from both basic biological and therapeutic standpoints. Our previous studies, based on mouse models, advanced the knowledge of the disease and the specific cell types involved in the RTT neuropathology. We showed that mutant glia, specifically astrocytes, are integral part of RTT and that healthy astrocytes are able to rescue many aspects of the disease. However, mouse models do not represent properly human RTT, which is known to be more severe than in mouse models. Importantly, human astrocytes are significantly different than mouse astrocytes, both in their structure and in the gene expression landscape. In the past decade, RTT patient-derived iPSCs have been used to generate neurons and glia in monolayer as a model for human RTT. While this model is useful, it does not reflect faithfully the generation and maturation of neurons and glia in the developing brain. We propose to generate 3-dimensional (3D) human cortical spheroids (hCSs) derived from human pluripotent stem cells (hPSCs) as a model for RTT brain development, in order to produce, isolate, and study mature astrocytes. We will exploit the advantages of the long-term 3D spheroid model, which allows structural, transcriptional and functional maturation of astrocytes, resembling the human postnatal maturation stage, which parallels the onset of RTT. We will determine how RTT-causing mutations in MECP2 affect the intrinsic properties of mature postnatal human astrocytes when they develop and mature with neighboring neurons, their ability to support neuronal structural plasticity, their gene expression profile, and their metabolic signatures. Modeling the development and maturation of human RTT astrocytes and analyzing their cellular and molecular properties at a stage which parallels the onset of RTT will provide important insights into the non-cell autonomous mechanism of RTT and serve as a platform for developing therapeutic strategies to reverse the impaired human RTT neuronal networks.