A current challenge in cancer therapeutics is incomplete response to treatment and emergence of therapy-resistant disease. Androgen deprivation therapy (ADT) is the standard treatment for advanced prostate cancer and effectively reduces the tumour burden in most patients. Yet, residual tumour cells that withstand ADT eventually develop lethal castration-resistance. Eliminating these castrate-tolerant cells, by combining ADT with other treatments, might delay or even prevent castration-resistant prostate cancer (CRPC). Clinical trial evidence supports this notion, with the CHAARTED and STAMPEDE studies showing that combining ADT with upfront docetaxel improves the overall survival of men with metastatic prostate cancer. These studies suggest that chemotherapeutics and other AR-targeted therapies target a population of cells that are not sensitive to castration alone. Therefore, we hypothesise that more effective co-targeting strategies could eliminate castrate-tolerant cells and improve outcomes for men with advanced prostate cancer. To identify signalling pathways that facilitate the survival of castrate-tolerant cells, we used prostate cancer patient-derived xenografts (PDXs) and single-cell transcriptomics. We show that a subpopulation of castrate-tolerant cells exist in multifocal regions of low, intermediate and high risk tumours, and can survive long-term castration. Castrate-tolerant cells significantly upregulate components of the retinoic acid signalling pathway, including CRABP2 (Cellular Retinoic Acid Binding Protein 2) and RARRES3 (Retinoic Acid Receptor Responder 3). Pre-clinical studies with PDX-derived organoids showed that inhibiting retinoic acid signalling stimulates the growth of castrate-tolerant cells and renders them sensitive to docetaxel treatment. Altogether, these data show that specific signalling pathways are up-regulated in castrate-tolerant cells, including retinoic acid signalling, providing rational co-targeting strategies to improve the efficacy of ADT and delay or prevent progression to CRPC.