FKBPL and its peptide derivative, AD-01, have already demonstrated well-established inhibitory effects on breast cancer growth and CD44 dependent anti-angiogenic activity. FKBPL stable overexpression or AD-O! treatment were highly effective at reducing the BCSC population measured by inhibiting mammosphere forming efficiency (MFE) in cell lines and primary breast cancer samples from both solid breast tumours and pleural effusions. Flow cytometry, to assess the ESA+/CD44+/CD24' subpopulation, validated these results. The ability of AD-01 to inhibit the selfrenewal capacity of BCSCs was confirmed across three generations of mammospheres, where mammospheres were completely eradicated by the third generation. Clonogenic assays suggested that AD-O! mediated BCSC differentiation, with a significant decrease in the number of holoclones and an associated increase in meroclones/paraclones. In support of this, the stem cell markers, Nanog, Oct4 and Sox2 were significantly reduced following AD-01 treatment, whilst transfection of FKBPL-targeted siRNAs led to an increase in these markers and in mammosphere forming potential, highlighting the endogenous role of FKBPL in BCSC signalling. The clinical relevance of this was confirmed using a publically available microarray data set (GSE7390), where high FKBPL and low Nanog expression were independently associated with improved overall survival in breast cancer patients. When AD-01 was combined with other agents, we observed additive activity with the Notch inhibitor, DAPT and AD-Ol was also able to abrogate a chemo- and radiotherapy induced enrichment in BCSCs. Importantly, using 'gold standard' in vivo limiting dilution assays we demonstrated a delay in tumour initiation and reoccurrence in AD-O! treated xenografts. The anti-BCSC mechanism of action of the FKBPL endogenous protein and its peptide derivative, AD-01, involves the CD44 and Notch pathway. In summary, AD-Ol appears to have dual anti-angiogenic and anti-BCSC activity which will be advantageous as this agent enters clinical trial.