Hematopoietic stem cells (HSCs) have been studied for decades in order to understand their stem cell biology and their potential as treatments in gene therapy, and those studies have resulted in tremendous advancement of understanding HSCs. However, most of the studies required the sacrifice of cohorts of the animals in order to obtain data for analysis, resulting in the use of large animal numbers along with difficult long-term studies. The dynamic engraftment and expansion of HSC are not fully observed and analyzed. Until recently, with the development of optical imaging, HSC repopulation can be continuously monitored in the same animal over a long period of time, reducing animal numbers and opening a new dimension for investigation. In this chapter, bioluminescence imaging of murine HSC is described for observing the dynamic repopulation process after transplantation. Photons emitted from transplanted murine HSCs expressing firefly luciferase within the mice can be visualized in light-sealed chamber with a highly sensitive digital camera after injection of substrate D-luciferin. Xenogen IVIS200 imaging system is used to record the process, and other similar imaging systems can also be used for this process.