Tracking the gene expression programs and clonal relationships that underlie mast, myeloid, and T lineage specification from stem cells

T cells develop from hematopoietic progenitors in the thymus and protect against pathogens and cancer. However, the emergence of human T cell-competent blood progenitors and their subsequent specification to the T lineage have been challenging to capture in real time. Here, we leveraged a pluripotent stem cell differentiation system to understand the transcriptional dynamics and cell fate restriction events that underlie this critical developmental process. Time-resolved single-cell RNA sequencing revealed that downregulation of the multipotent hematopoietic program, upregulation of >90 lineage-associated transcription factors, and cell-cycle exit all occur within a highly coordinated developmental window. Gene-regulatory network inference uncovered a role for YBX1 in T lineage specification. We mapped the differentiation cell fate hierarchy using transcribed lineage barcoding and discovered that mast and myeloid potential bifurcate from each other early in hematopoiesis, upstream of T lineage restriction. Our systems-level analyses provide a quantitative, time-resolved model of human T cell fate specification. A record of this paper’s transparent peer review process is included in the supplemental information. •Pluripotent stem cells are a resource for studying human immune fate specification•Knocking out YBX1 reduces T lineage output•Mast potential and myeloid potential diverge early during blood development•T cells and myeloid cells have a high degree of shared clonal ancestry Michaels et al. use in vitro differentiation to analyze the gene expression programs and fate hierarchy that underlie human mast, myeloid, and T lineage specification with fine temporal and clonal resolution. They show that YBX1 regulates T cell potential and that mast and myeloid fates diverge early during blood development.