Even though hematopoietic stem cells (HSC) are amongst the first somatic stem cells exploited for therapeutic purposes, their application is still limited by the inability to expand them ex vivo without impairing their function. Moreover, it has recently emerged that several types of leukemia develop and relapse through complex interactions with bone marrow (BM) components and may directly affect the HSC and their niche. Increasing attention has therefore been dedicated to the BM microenvironment the HSC reside in, with the view that a better understanding of the molecular regulators of HSC-niche interaction in vivo will allow improving HSC mobilization, collection and transplantation and provide clues for the development of innovative leukemia treatments. This chapter focuses on a recently established technique for the visualization of transplanted hematopoietic stem and progenitor cells (HSPC) within the calvarium bone marrow of live mice (Lo Celso et al. Nature 457:92-96, 2007). Intravital microscopy is a rapidly developing field, driven by constant improvement in both detection technologies (i.e., spatial resolution, depth of penetration, spectral definition) and probe availability (i.e., increasingly sophisticated genetic and chemical reporter systems). We therefore discuss the current limitations and challenges related to intravital microscopy of the HSC niche and introduce a number of potential imaging approaches, which could be promising candidates for future development of this technique.