background: Previous research in FMS-like tyrosine kinase 3 ligands (FLT3L) has primarily focused on their potential to generate dendritic cells (DCs) from bone marrow progenitors, with a limited understanding of how these cells affect CD8 T cell function. In this study, we further investigated the in vivo role of FLT3L for the immunomodulatory capabilities of CD8 T cells. methods: Albumin-conjugated FLT3L (Alb-FLT3L) was generated and applied for translational medicine purposes; here it was used to treat naïve C57BL/6 and OT1 mice for CD8 T cell response analysis. Syngeneic B16ova and E.G7ova mouse models were employed for adoptive cell transfer to evaluate the effects of Alb-FLT3L preconditioning of CD8 T cells on tumor progression. To uncover the underlying mechanisms of Alb-FLT3L modulation, we conducted bulk RNA-seq analysis of the CD44 CD8 T cells. STAT1-deficient mice were used to elucidate the functional roles of Alb-FLT3L in the modulation of T cells. Finally, antibody blockade of type one interferon signaling and in vitro coculture of plasmacytoid DCs (pDCs) with naive CD8 T cells was performed to determine the role of pDCs in mediating regulation of CD44 CD8 T cells. results: CD44 CD8 T cells were enhanced in C57BL/6 mice administrated with Alb-FLT3L. These CD8 T cells exhibited virtual memory features and had greater proliferative and effective functions. Notably, the adoptive transfer of CD44 naïve CD8 T cells into C57BL/6 mice with B16ova tumors led to significant tumor regression. RNA-seq analysis of the CD44 naïve CD8 T cells revealed FLT3L to induce CD44 CD8 T cells in a JAK-STAT1 signaling pathway-dependent manner, as supported by results indicating a decreased ability of FLT3L to enhance CD8 T cell proliferation in STAT1-deficient mice as compared to wild-type control mice. Moreover, antibody blockade of type one interferon signaling restricted the generation of FLT3L-induced CD44 CD8 T cells, while CD44 expression was able to be induced in naïve CD8 T cells cocultured with pDCs derived from FLT3L-treated mice. This suggests the crucial role of pDCs in mediating FLT3L regulation of CD44 CD8 T cells. conclusions: These findings provide critical insight and support the therapeutic potential of Alb-FLT3L as an immune modulator in preconditioning of naïve CD8 T cells for cancer immunotherapy.