Birt-Hogg-Dubé (BHD) syndrome is associated with cyst formation in the lung but the link with the mutations in the folliculin gene which cause this disease remains unknown. Turnover of the airway epithelium is maintained by the autocrine action of epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF) which induce clearance of aged cells and promote re-growth from a progenitor cells. However, culture models lacking Flcn display slower growth and form poor cell junctions suggesting that the process of lung cyst formation could arise from a defect in this process. We therefore tested the hypothesis that Flcn alters growth responses to autocrine signals which regulate airway epithelial growth. Flcn expression levels were manipulated in an immortalised human bronchial epithelial cell line (16HBE14o- or “HBE”), by: i) Flcn over-expression, iii) transient siRNA or iii) stable shRNA knockdown. Comparisons were made with similarly treated control cells respectively transformed with empty or non-target constructs. Phosphorylation (T202/Y204) of ERK1/2 was used to indicate growth factor receptor activity and ELISA was used to assess growth factor secretion. Both transient and stable knock-down methods resulted in ~60% reduction in Flcn levels compared to controls and fluorescence-activated cell sorting (FACS) analysis revealed that this loss induced cell accumulation in G0/G1 phases of the cell cycle with a corresponding decline in pre-mitotic S phase cells. Recombinant EGF (10ng.ml-1.6hrs) did not alter ERK1/2 phosphorylation on either Flcn over-expressing or knockdown HBE but raised VEGF secretion from 80.8±6.0 to 184.4±14.3pg.mgP-1 suggesting that its major effect is to induce VEGF signalling. Addition of recombinant VEGF (10ng.ml-1.6hrs) to control and shRNA Flcn knock-down cells raised ERK1/2 and PI3-kinase (phosphoAKTT308) activity in both cell lines, however, the rate, magnitude and duration these effects were each suppressed in Flcn shRNA cells (P<0.05; n=4). We examined VEGF receptor 2 (VEGFR2) mRNA and protein expression and found that both were elevated by ~1.7 fold in Flcn shRNA cells. However, recombinant VEGF failed to induce receptor phosphorylation at Y1175 in these cells, a critical residue for receptor internalisation and activation (n=4). This was supported by immunofluorescence which revealed that VEGFR2 remained membrane-associated onVEGF treatment and failed to form active endosomal complexes. We conclude that Flcn knockdown suppresses autocrine signalling via the VEGF pathway by altering VEGFR2 signalling. Given the key role played by this factor in maintaining airway epithelial integrity, its dysregulation by Flcn mutation is likely to be a contributing factor for lung cyst formation in the BHD lung.