Under stress hematopoiesis, previous studies have suggested the migration of hematopoietic stem cells (HSCs) from bone marrow (BM) to extramedullary sites such as the spleen. However, there is little direct evidence of HSC migration from the BM to the spleen. Here, we induced myeloablation via 5-fluorouracil (5-FU) and showed direct evidence of HSC migration from BM to spleen during hematopoietic regeneration via a photoconvertible fluorophore. Moreover, during steady state, HSCs preferentially migrated to BM rather than spleen, but during hematopoietic regeneration, HSCs preferred spleen as a migration site equivalently or greater. Furthermore, in the early phase, HSCs egressed from BM through the attenuated HSC retention. However, HSCs in the late phase gained significantly enhanced cell-autonomous motility, which was independent of chemotaxis. Collectively, HSC mobilization from BM, before the migration to the spleen, was dynamically changed from passive to active events during hematopoietic regeneration.