Hematopoietic stem cell transplantation (HSCT) is a groundbreaking therapy that has transformed the treatment of severe hematological and immune disorders. It involves infusing healthy or corrected hematopoietic stem cells (HSCs) into a patient to restore their blood cell production. Despite its remarkable success, HSCT is currently limited to the most severe cases due to several key challenges. These bottlenecks include inefficient HSC mobilization, limited engraftment leading to insufficient clonal diversity, and the toxicity of chemotherapy-based conditioning regimens. This comprehensive proposal outlines a transformative approach to address these limitations head-on, enhancing both the efficacy and safety of HSCT. The project aims to: 1. Uncover the key factors governing HSC engraftment and egress: Using optimized genome-wide gain- and loss-of-function in vivo screening in HSCs, I will identify the pivotal players responsible for HSC engraftment and egress. These insights will inform the development of innovative mobilization strategies and the engineering of HSCs to confer a transient engraftment advantage. 2. Establish a mobilization-based conditioning regimen: I will exploit the temporary niche depletion induced by mobilizers to optimize a genotoxic-free regimen. By harnessing cell surface profiling data and potentially using mobilizer-resistant variants generated through directed evolution, I will tilt the competitive balance between freshly mobilized and infused HSCs, favoring the engraftment of infused cells. This project seeks to pioneer avant-garde methods that enhance mobilization and engraftment while avoiding the use of toxic conditioning regimens, thereby addressing major HSCT limitations. These advancements hold the potential to transform HSCT into a safer, more broadly applicable therapy, extending its benefits to a wider demographic and diverse healthcare settings. This aligns with the ultimate objective of achieving global health equity.