In this proposal we show the preliminary development of a novel intravital methods to concurrently visualize both splenic colony formation and bone marrow reconstitution by fluorescently tagged donor HSC/HPC populations. Our tibia window model allows direct visualization of early marrow engraftment dynamics of fluorescently tagged HSC/HPC populations in individual living mice for up to three weeks. The intravital CFU- Spleen model currently allows 1-2 concurrent observations of the extramedullary splenic microenvironments and bone marrow longitudinally in individual transplant recipients. The primary goal of this SHINE-II proposal is to improve the intravital CFU-Spleen model to increase the number and resolution of the spleen observations to better match the tibia window model. The ability to observe both marrow and extramedullary donor HSC/HPC engraftment and niche utilization dynamics in living recipients over multiple days/weeks post transplant will significantly enhance our understanding of transplant biology.