A coordinated transcriptional network accompanied NK cell cytotoxic differentiation trajectory diverged from non-cytotoxic ILC identities. (A) Projections of RNA velocity vectors on the CD56⁺ cell UMAP described the 3 major cellular trajectories governed by EOMES, HOBIT, and RORC. (B) RNA velocity latent-time displayed as color scale, indicates suggested regions of root cells (top-right) and end points (for NK trajectory; bottom-right). (C) Expression levels of progenitor-associated genes: MYC, TCF7, BACH2, and α4β7 integrin. (D) Heat map shows dynamic expression of early and late-acting TFs with cells aligned according to NK cell cytotoxic trajectory. (E) Heat map showing selected top 20 master TFs associated with highly dynamic regulons identified via SCENIC, aligned following the RNA velocity latent-time of NK cell trajectory as in panel D. Custom annotation columns indicated: (i) number of genes associated with each regulon (bar plot), (ii) average regulon activity score (area under the curve [AUC] score) respective for each regulon detected (violin plot). (F) Regulon activity (AUC score) among the top 3 regulons: EOMES, IKZF3, and CEBPD as key drivers along the cytotoxic NK cell trajectory (red) compared with alternative trajectory (tissue resident–like NK cell; blue). (G) Network of coordinated genes governed by EOMES identified via SCENIC and the corresponding biological pathways highly enriched for EOMES-driven network.