Rett syndrome (RTT) is a debilitating neurodevelopmental disorder caused by mutations in theMECP2gene. To facilitate the study of cellular mechanisms in human cells, we established several human stem cell lines: human embryonic stem cell (hESC) line carrying the common T158M mutation (MECP2^T158M/T158M), hESC line expressing no MECP2 (MECP2-KO), congenic pair of wild-type and mutant RTT patient-specific induced pluripotent stem cell (iPSC) line carrying the V247fs mutation (V247fs-WT and V247fs-MT), and iPSC line in which the V247fs mutation was corrected by CRISPR/Cas9-based genome editing (V247fs-MT-correction). Detailed analyses of forebrain neurons differentiated from these human stem cell lines revealed genotype-dependent quantitative phenotypes in neurite growth, dendritic complexity, and mitochondrial function. At the molecular level, we found a significant reduction in the level of CREB and phosphorylated CREB in forebrain neurons differentiated fromMECP2^T158M/T158M,MECP2-KO, and V247fs-MT stem cell lines. Importantly, overexpression of CREB or pharmacological activation of CREB signaling in those forebrain neurons rescued the phenotypes in neurite growth, dendritic complexity, and mitochondrial function. Finally, pharmacological activation of CREB in the femaleMecp2heterozygous mice rescued several behavioral defects. Together, our study establishes a robustin vitroplatform for consistent quantitative evaluation of genotype-dependent RTT phenotypes, reveals a previously unappreciated role of CREB signaling in RTT pathogenesis, and identifies a potential therapeutic target for RTT.SIGNIFICANCE STATEMENTOur study establishes a robust human stem cell-based platform for consistent quantitative evaluation of genotype-dependent Rett syndrome (RTT) phenotypes at the cellular level. By providing the first evidence that enhancing cAMP response element binding protein signaling can alleviate RTT phenotypes bothin vitroandin vivo, we reveal a previously unappreciated role of cAMP response element binding protein signaling in RTT pathogenesis, and identify a potential therapeutic target for RTT.