Bioluminescence imaging enables the real-time detection and tracking of engrafted cells in vivo noninvasively and dynamically. By detecting and quantifying the photons released from the oxidation of luciferin catalyzed by luciferase enzymes, this approach has proven effective in tracking engrafted stem cell survival and retention, making it a powerful tool to monitor cell fate after transplantation without animal sacrifice. Here we describe a protocol that allows luciferase-labeled stem cells to be imaged and tracked in vivo by bioluminescent imaging via an IVIS spectrum imaging system.