Cancer stem cells (CSCs) are a highly aggressive and therapy-resistant tumor subpopulation that is a significant contributing factor to cancer treatment failure. Synthetic biology equips researchers with advanced platforms to interrogate, engineer, and control CSC behavior. Despite its potential, the application of synthetic biology within the CSC field remains in its infancy, as progress is hindered by the current lack of precise, scalable approaches to effectively manipulate CSC behavior. This is compounded by challenges including high degree of CSC heterogeneity, limited understanding of their microenvironmental interactions, and the difficulty of engineering synthetic circuits that can operate consistently in the complex and evolving context of cancer. This chapter explores the role of synthetic and nanotechnology applications in CSC regulation and discusses synthetic biology approaches including top-down and bottom-up modulation for functionality and de novo synthesis. Next, synthetic biology and nanotechnology-based combination therapies for CSCs are examined, including engineered gene networks, clustered regularly interspaced short palindromic repeats platform, RNA-based systems, and nanotechnology. Finally, the discussion concludes with current challenges and future directions for advancing synthetic biology-based interventions in CSC research.