Cancer stem cells (CSCs) are a subpopulation of tumour cells endowed with self-renewal and multi-lineage differentiation capacity. CSCs are highly tumorigenic and are proposed to drive cancer relapse and metastasis. We are examining CSC heterogeneity, including which CSC markers/properties most correlate with self-renewal capacity and tumorigenicity. Emerging literature suggests that embryonic stem cell pluripotency genes are critical controllers of CSC self-renewal, and that the self-renewing population is slow cycling relative to the tumour bulk. To identify a CSC population with high pluripotency gene expression, we established breast cancer cell lines carrying the Oct4 promoter driving green fluorescent protein (Oct4-GFP); FACS-isolated Oct4-GFPbright cells were highly enriched for endogenous Oct4 and Sox2 expression. To identify a slow cycling cancer cell population, a label retention assay using the VPD450 proliferation dye was established. Using spectrally compatible flurophores, we simultaneously assessed Oct4-GFP expression, VPD450 label retention, and surface antigen profile. We find considerable overlap between slow cycling and pluripotency gene-expressing cells, but less overlap of these populations with cells expressing conventional breast CSC surface markers (e.g. CD44/CD24/ESA). Ongoing work is defining relative tumorigenicity of these populations, and the effect of manipulating pluripotency gene expression on tumorigenicity, self-renewal, and cycling rate. In addition to probing the functions of pluripotency genes in CSCs, we are using our reporter lines as tools to screen for novel anti-CSC agents, as well as characterizing transcriptional and epigenetic pathways controlling CSCs. In particular, we have determined that the HDAC inhibitor Valproic acid (VPA) strongly induces pluripotency gene expression in breast CSC and increases the slow cycling population. VPA is proposed as a chemotherapy adjuvant to improve response to hormonal therapy in breast cancer. Our data indicates that VPA may also drive breast CSC self-renewal underscores the need to assess the differential effects of drugs on bulk tumour cells and CSC.