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Vertebrate evolution was accompanied by two rounds of whole-genome duplication followed by functional divergence in terms of regulatory circuits and gene expression patterns. As a basal and slow-evolving chordate species, amphioxus is an ideal paradigm for exploring the origin and evolution of vertebrates. Single-cell sequencing has been widely used to construct the developmental cell atlas of several representative species of vertebrates (human, mouse, zebrafish, and frog) and tunicates (sea squirts). Here, we perform single-nucleus RNA sequencing (snRNA-seq) and single-cell assay for transposase accessible chromatin sequencing (scATAC-seq) for different stages of amphioxus (covering embryogenesis and adult tissues). With the datasets generated, we constructed a developmental tree for amphioxus cell fate commitment and lineage specification and characterize the underlying key regulators and genetic regulatory networks. The data are publicly available on the online platform AmphioxusAtlas. [Display omitted] • A snRNA-seq atlas of amphioxus developmental embryos and multiple adult tissues • A scATAC-seq atlas of amphioxus developmental embryos • A developmental tree for amphioxus cell fate commitment and lineage specification • Computational identification and validation of lineage-specific markers Ma et al. conduct a joint profiling of gene expression and chromatin accessibility during amphioxus embryogenesis at single-cell resolution. With the datasets generated, a developmental tree for amphioxus cell fate commitment and lineage specification is constructed, and multiple markers for each lineage are identified and validated. [ABSTRACT FROM AUTHOR]