Breast cancer stem cell (BCSC) activity is enhanced following anti-estrogen treatment of Estrogen Receptor positive (ER+) breast cancer, leading to endocrine therapy resistance. Notch4 receptor signalling is highly activated in these BCSCs, linking Notch4 activity to endocrine therapy resistance (Simoes, O'Brien, et al., 2015). In Drosophila, the Notch AxE2 mutant is involved in ligand-independent, Deltex-dependent signalling (Shimizu et al., 2014). Human Notch4 possesses the equivalent residue change to this mutant (Y914), diverging from Notch1/2/3. It is therefore hypothesised that Notch4 may signal in a Deltex-dependent or ligand-independent manner, increase BCSC activity and drive endocrine resistance. Notch4 mutants selected during endocrine therapy may further increase Deltex-dependent signalling. Immunofluorescence and gene expression analysis showed that Notch4 signals via an endocytic pathway mechanism in breast cancer cells, passing through endosomes and lysosomes as a full length receptor for activation. We found that Deltex1, Deltex4 and Rab7a are specifically involved in this signalling pathway. Deltex4 is essential for BCSC activity of Notch4 cells and TRPML (facilitates fusion of endosomes and lysosomes) was found to be required for viability and BCSC activity of endocrine resistant and Notch4 reliant cells, whilst ADAM10 (undertakes a cleavage of Notch in the canonical signalling pathway) was not. Creation of a stable Notch4 overexpressing cell line demonstrated that Notch4 increases Hey2 expression preferentially over the other Notch target genes Hes1 and Hey1. This Notch4 cell line was found to have increased Notch signalling, BCSC activity and resistance to endocrine therapy. Analysis of breast cancer genomic databases identified increased numbers of Notch4 mutations in metastatic breast cancers compared to primary breast cancers. Investigating selected breast cancer associated Notch4 mutations using site-directed mutagenesis identified unique signalling and trafficking roles. Through its reversion to the residue found in Notch1/2/3, the Y914 Notch4 residue was discovered to be required for Notch4 endocytic trafficking and BCSC activity. In conclusion, we have shown that Notch4 can signal via an endocytic pathway route, which requires Deltex. Deltex4 and TRPML are required for Notch4 mediated/ endocrine resistant BCSC activity. These actions are linked to the Notch4 Y914 residue which has been linked to ligand independent Notch activation in Drosophila. Using knowledge gained about Notch4 signalling mechanisms in BCSCs, the future aim is identification of therapeutic targets to reduce Notch4 activity and treat breast cancer stem cells, to be used in combination with endocrine therapy.