Abstract In this study we set out to define the prominent fibroblast subpopulations found in human non-small cell lung cancer. Fibroblasts are functionally heterogeneous cells, capable of promoting and suppressing tumor progression. Across cancer types, the extent and cause of this phenotypic diversity remains unknown. We used single-cell RNA sequencing and multiplexed immunohistochemistry to examine fibroblast heterogeneity in human lung and non-small cell lung cancer (NSCLC) samples. We then performed in silico and in vitro analyses to examine the molecular mechanisms regulating fibroblast phenotypes. Single-cell RNA sequencing identified seven fibroblast subpopulations: including inflammatory fibroblasts and myofibroblasts (representing terminal differentiation states), quiescent fibroblasts, proto-myofibroblasts (x2) and proto-inflammatory fibroblasts (x2). Multiplex immunohistochemistry showed that these fibroblast subpopulations were variably distributed throughout tissues but accumulated at discrete niches associated with differentiation status. Bioinformatics analyses suggested TGF-β1 and IL-1 as key regulators of myofibroblastic and inflammatory differentiation respectively. However, in vitro analyses showed that whilst TGF-β1 stimulation in combination with increased tissue tension could induce myofibroblast marker expression, it failed to fully re-capitulate ex-vivo¬ phenotypes. Similarly, IL-1β treatment only induced upregulation of a subset of inflammatory fibroblast marker genes. In silico modelling of ligand-receptor signaling identified additional pathways and cell interactions likely to be involved in fibroblast activation. This highlighted a potential role for IL-11 and IL-6 (among other ligands) in myofibroblast and inflammatory fibroblast activation respectively. This analysis provides valuable insight into fibroblast subtypes and differentiation mechanisms in NSCLC. Citation Format: Christopher Jon Hanley, Sara Waise, Parker Rachel, Maria-Antoinette Lopez, Julian Taylor, Lucy Kimbley, Jonathon West, Christian Ottensmeier, Mat Rose-Zerilli, Gareth Thomas. Spatially discrete signalling niches regulate fibroblast heterogeneity in human lung cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PO-039.