A desperate need exists to develop technology to regenerate sperm that can be used for in vitro fertilization (IVF) among men who lack sperm production, such as pediatric cancer survivors. In Canada, approximately 2,440 boys aged less than 15 will be diagnosed with cancer each year. Fortunately, the field of oncology has made significant improvements in survival rates, which are estimated to be 83%. However, treatments will render up to 97% of paediatric cancer survivors infertile with no sperm production, despite over 75% eventually desiring to have biological children. While stem cells (sperm precursors) can be retrieved prior to cancer therapies, no technology currently exists to regenerate sperm, which is required to achieve a pregnancy. This project proposes to utilize single cell sequencing and along with state-of-the-art computational modelling to reveal molecules and pathways that are key regulators of developing sperm from stem cells. These findings will be screened and tested to identify critical molecules that help generate sperm in 3D bioprinted structures. Results from this study will contribute to developing the understanding and technology to regenerate sperm for men lacking any ability to father biological children.