The blood-brain barrier (BBB) is essential for maintaining the homeostasis of the central nervous system. However, the processes of BBB formation in humans remain unclear. Here, using single-cell spatiotemporal transcriptomics, we investigate human BBB development from 6 to 21 gestational weeks (GWs) and observe widespread expression of BBB-specific transporters in all brain-endothelial subclusters during development. We determine the onset of the human BBB-like transcriptional signature at GW8 and prove that neural cells can induce the expression of BBB-specific transporters in brain endothelial cells (ECs) via CADHERIN-2 (CDH2). We also demonstrate that neural progenitor cells promote the proliferation of mural cells. Concomitant with the initiation of the BBB-like transcriptional signature, communication signals between ECs and mural cells begin to intensify. In addition, we reveal conserved BBB development between humans and mice and demonstrate that H2A.Z.1 regulates angiogenesis and BBB development. Collectively, these findings provide unique insights into understanding human BBB ontogeny and identifying therapeutic targets for BBB-related disorders.