The human cerebral cortex is composed of six layers and dozens of areas that are molecularly and structurally distinct1–4. Although single-cell transcriptomic studies have advanced the molecular characterization of human cortical development, a substantial gap exists owing to the loss of spatial context during cell dissociation5–8. Here we used multiplexed error-robust fluorescence in situ hybridization (MERFISH)9, augmented with deep-learning-based nucleus segmentation, to examine the molecular, cellular and cytoarchitectural development of the human fetal cortex with spatially resolved single-cell resolution. Our extensive spatial atlas, encompassing more than 18 million single cells, spans eight cortical areas across seven developmental time points. We uncovered the early establishment of the six-layer structure, identifiable by the laminar distribution of excitatory neuron subtypes, 3 months before the emergence of cytoarchitectural layers. Notably, we discovered two distinct modes of cortical areal specification during mid-gestation: (1) a continuous, gradual transition observed across most cortical areas along the anterior–posterior axis and (2) a discrete, abrupt boundary specifically identified between the primary (V1) and secondary (V2) visual cortices as early as gestational week 20. This sharp binary transition in V1–V2 neuronal subtypes challenges the notion that mid-gestation cortical arealization involves only gradient-like transitions6,10. Furthermore, integrating single-nucleus RNA sequencing with MERFISH revealed an early upregulation of synaptogenesis in V1-specific layer 4 neurons. Collectively, our findings underscore the crucial role of spatial relationships in determining the molecular specification of cortical layers and areas. This study establishes a spatially resolved single-cell analysis paradigm and paves the way for the construction of a comprehensive developmental atlas of the human brain.
