show Abstracthide AbstractInnate lymphoid cells (ILCs) play strategic roles in tissue homeostasis and immunity. ILCs arise from lymphoid progenitors undergoing lineage restriction leading to the development of specialised ILC subsets. We generated '5x polychromILC' compound transcription factor reporter mice to delineate ILC precursor states by revealing the multifaceted expression of key ILC-associated transcription factors (Id2, Bcl11b, Gata3, Rorc(?t) and Rora) during ILC development in the bone marrow. This approach allowed previously unattained enrichment of rare progenitor subsets and revealed hitherto unappreciated ILC precursor heterogeneity. In vivo and in vitro assays identified novel precursors with potential to generate all ILC subsets and natural killer cells, and also permitted discrimination of elusive ILC3 bone marrow antecedents. Single cell gene expression analysis identified a discrete ILC2-committed population and delineated transition states between early progenitors and a highly heterogeneous ILC1/3/NK precursor cell cluster. This diversity may facilitate greater lineage potential upon progenitor recruitment to peripheral tissues. Overall design: Bone marrow was removed from femurs, tibiae and hips by flushing with PBS + 2% FCS, or by centrifuging briefly at 6000 x g. Red blood cells were removed by incubation with RBC lysis solution (140 mM NH4Cl, 17 mM Tris; pH 7.2). Single cell suspensions were incubated with fluorochrome-, or biotin-, conjugated antibodies in the presence of anti-CD16/CD32 antibody (Fc block, clone 2.4G2), followed by fluorochrome-conjugated streptavidin. We purified individual Lin–Id2+ bone marrow cells (a small number of Lin–Id2– cells were also sampled for comparison) and performed single cell (sc)RNA-seq to profile progenitor cell gene expression. Subsequently, in order to obtain sufficient representation of the less frequent subsets we purified bone marrow progenitors based on the transcription factor and surface marker expression profiles that defined the subpopulations outlined above and summarised in Table 1.