Data Description Stitched & rotated DAPI images - tiff file format filename indicates sample and optical z-slice position, i.e. embryo3_z5.tif is the DAPI image for embryo 3 in optical z-slice 5. Also provided in PNG format. Detected molecules and cell segmentation in MoleculeExperiment objects - RDS files to read data using the MoleculeExperiment format in R/Bioconductor. Filename embryo3_z5.Rds indicates MoleculeExperiment RDS file for embryo 3 in optical z-slice 5. Coordinates are provided in microns. Note that z-slices 2 and 5 are only provided for embryos 1,2,3 as they were originally provided in Lohoff et al, Nature Biotechnology, 2023. Pixels-to-microns conversion - pixelSize.R Simple R script/text to indicate the size of each pixel in the DAPI images, this is to align the coordinate systems between the DAPI images and molecules. Project Abstract At the onset of murine gastrulation, pluripotent epiblast cells migrate through the primitive streak, generating mesodermal and endodermal precursors, while the ectoderm arises from the remaining epiblast. Together, these germ layers establish the body plan, defining major body axes and initiating organogenesis. Although comprehensive single cell transcriptional atlases of dissociated mouse embryos across embryonic stages have provided valuable insights during gastrulation, the spatial context for cell differentiation and tissue patterning remain underexplored. In this study, we employed spatial transcriptomics to measure gene expression in mouse embryos at E6.5 and E7.5 and integrated these datasets with previously published E8.5 spatial transcriptomics and a scRNA-seq atlas spanning E6.5 to E9.5. This approach resulted in a comprehensive spatiotemporal atlas, comprising over 150,000 cells with 88 refined cell type annotations as well as genome-wide transcriptional imputation during mouse gastrulation and early organogenesis. The atlas facilitates exploration of gene expression dynamics along anterior-posterior and dorsal-ventral axes at cell type, tissue, and organismal scales, revealing insights into mesodermal fate decisions within the primitive streak. Moreover, we developed a bioinformatics pipeline to project additional scRNA-seq datasets into a spatiotemporal framework and demonstrate its utility by analysing cardiovascular models of gastrulation3. To maximise impact, the atlas is publicly accessible via a user-friendly web portal empowering the wider developmental and stem cell biology communities to explore mechanisms of early mouse development in a spatiotemporal context.