The adult human brain comprises more than a thousand distinct neuronal and glial cell types, a diversity that emerges during early brain development. To reveal the precise sequence of events during early brain development, we used single-cell RNA sequencing and spatial transcriptomics and uncovered cell states and trajectories in human brains at 5 to 14 postconceptional weeks (pcw). We identified 12 major classes that are organized as ~600 distinct cell states, which map to precise spatial anatomical domains at 5 pcw. We described detailed differentiation trajectories of the human forebrain and midbrain and found a large number of region-specific glioblasts that mature into distinct pre-astrocytes and pre–oligodendrocyte precursor cells. Our findings reveal the establishment of cell types during the first trimester of human brain development. INTRODUCTION The adult human brain is divided into hundreds of spatial domains, each comprising tens or hundreds of distinct neuronal, glial, and other cell types. This complex arrangement of cells is initially established during the first trimester of development, yet the difficulty of accessing such early embryos has hindered detailed molecular analysis. Dissecting the spatial, temporal, and transcriptional changes that occur in the whole brain during the first trimester promises to reveal the fundamental blueprint of the human brain. RATIONALE To comprehensively map brain cell types and gene expression trajectories during the first trimester, we collected 26 brain specimens spanning 5 to 14 postconceptional weeks (pcw) that were dissected into 111 distinct biological samples. Each of these samples was subjected to single-cell RNA sequencing, resulting in a collection of 1,665,937 high-quality single-cell transcriptomes. These data were complemented by a spatial transcriptomic analysis at 5 pcw using highly multiplexed RNA fluorescence in situ hybridization (FISH) and spatial transcriptomics. We identified 616 clusters, which we annotated with metadata, including class and subclass, spatial location, embryonic age distribution, and specific gene expression markers. RESULTS The detailed resolution of the dataset allowed us to characterize general principles of brain development as well as delineate the differentiation trajectories of several brain regions. The developing excitatory neuron lineages in the neocortex revealed three different ongoing molecular programs: differentiation from radial glia to neurons, cell cycle, and maturation. We found a delicate balance between progenitor and differentiation factors in intermediate progenitor cells (IPCs), with the induction of neurogenic transcription factors visible after the G 1 cell cycle phase. Our findings support a conserved progressive transcriptional maturation in older specimens. Many genes were induced in late radial glia and glioblasts, making up a program that drives progenitors toward neurogenesis as well as gliogenesis. In the forebrain γ-aminobutyric acid–mediated (GABAergic) neuronal lineage, we found evidence of migration of CRABP -expressing cells from the medial ganglionic eminence into the thalamus, which are predicted to give rise to thalamic PVALB + neurons in the adult. Examining ventral midbrain development, we found a diverse set of progenitors already arising at 8 pcw, defining broad TH class identity, although adult TH subtype identities must arise after 14 pcw. Focusing on developing glia, we found a large set of region-specific glioblasts, of which most showed evidence of maturation into astrocytes. This provides a plausible mechanism for the specification of adult region-specific astrocyte types. We further identified oligodendrocyte precursor cells (OPCs) specific to the forebrain, midbrain, and hindbrain that expressed large numbers of functionally conserved genes. CONCLUSION Although previous studies have explored specific regions of the brain during development, this is the first known comprehensive study of the whole human brain during the crucial first trimester. We found that although neurons were the most diverse, both pre-astrocytes and OPCs were regionally distinct, and their gene expression suggests region- and cell type–specific supportive functions. These findings highlight the importance of early patterning events and provide a rich resource for the interpretation of the many brain disorders that show region-specific patterns of occurrence or severity and for identifying therapeutic targets for human disorders that affect specific brain cell populations. Atlas of the developing human brain. We studied the human brain using single-cell RNA sequencing from 5 to 14 pcw and multiplexed in situ RNA detection at 5 pcw. Our findings reveal how early patterning events establish the organization of the future brain and how maturation and differentiation trajectories are superimposed on this basic plan to generate the extraordinary complexity of the adult nervous system.