The Flt3-Flt3 ligand (Flt3L) pathway is critically involved in the differentiation and homeostasis of myeloid cells, including dendritic cells (DC); however, its role in the expansion and function of myeloid-derived suppressor cells (MDSC) has not been determined. In this article, we describe the ability of Flt3L to expand and activate murine MDSC capable of suppressing allograft rejection upon adoptive transfer. Although Flt3L expands and augments the stimulatory capacity of myeloid DC, MDSC expanded by Flt3L have increased suppressive activity. Although STAT3 is considered the central transcription factor for MDSC expansion, inhibition and genetic ablation of STAT3 did not block, but rather augmented, Flt3L-mediated MDSC expansion. MDSC suppressive function, preserved when STAT3 inhibition was removed, was reduced by genetic STAT3 deletion. Both STAT3 inhibition and deletion reduced Flt3L-mediated DC expansion, signifying that STAT3 had reciprocal effects on suppressive MDSC and immunostimulatory DC expansion. Together, these findings enhance our understanding of the immunomodulatory properties of Flt3L.