Rett Syndrome (RTT) is a X-linked neurodevelopmental disorder that predominantly affects girls. The detailed disease mechanisms remain unclear and fundamental treatments are yet to be established. The majority of typical RTT cases is triggered by mutations in the methyl CpG-binding protein 2 (MeCP2) gene. For the analyze of the disease mechanisms and drug development, in vitro models based on iPSC-derived cells with diseased MeCP2 are being investigated. In addition to MeCP2 mutations RTT is also affected by the genetic background, and thus it is necessary to analyze the effect of MeCP2 modifications in cells generated from multiple iPSC strains. Although the genome editing technology could provide such cellular models, the method tends to be time-consuming and costly. We therefore developed an in vitro RTT model using shRNA knockdown of MeCP2. iPSC-derived neurons and primary astrocytes were co-cultured and transduced with lentiviral vectors encoding MeCP2 shRNA. The neurons in the co-culture exhibited neurite atrophy defined as the reduction in the complexity of neural arborization. This neurite atrophy was rescued by overexpression of MeCp2, while the phenotype was absent in co-cultures transduced with control shRNA. Furthermore, brain-derived neurotrophic factor (BDNF), an enhancer of neurite arborization in RTT, significantly increased the neurite density in the MeCP2 knockdown co-cultures. These results suggests that the RTT model exhibits a disease-related neurite phenotype. Because the method can be easily applied to multiple iPSC strains, it is suited to generate RTT models with various genetic backgrounds and will contribute to the RTT research and high-throughput drug screening.