Flt3 ligand (FL) enhances hematopoietic cell proliferation and facilitates hematopoietic stem cell mobilization in vivo, while the stromal-derived factor 1alpha (SDF-1alpha, CXC ligand 12 [CXCL12])/CXC receptor 4 (CXCR4) axis is critical for their homing and trafficking. We investigated if FL and its receptor, Flt3, functionally interact with CXCL12/CXCR4 to regulate hematopoietic cell migration. FL stimulated chemokinetic activity when used alone, but synergistically enhanced short-term migration of CD34+ cells, Ba/F3 cells expressing human Flt3 (Ba/F3-Flt3), and human RS4;11 acute leukemia cells, induced by CXCL12. Moreover, overexpression of constitutively activated internal tandem duplication (ITD)-Flt3 mutants in Ba/F3 cells dramatically enhanced migration toward CXCL12. In Ba/F3-Flt3 cells, synergistic cell migration to FL plus CXCL12 was associated with enhanced phosphorylation of mitogen-activated protein kinase p42/p44 (MAPK(p42/p44)), cyclic adenosine monophosphate response element binding protein (CREB), and Akt, and was partially inhibited by pretreatment of cells with selective inhibitors for MAPK(p42/p44), protein kinase A (PKA), or phosphatidylinositol 3-kinase (PI3-kinase), implicating these pathways in migration to FL plus CXCL12. In contrast, prolonged exposure of CD34+ or Ba/F3-Flt3 cells to FL down-regulated CXCR4 expression, inhibited CXCL12-mediated phosphorylation of MAPK(p42/p44), CREB, and Akt, and impaired migration toward CXCL12. These findings suggest that FL/Flt3 may facilitate hematopoietic cell migration/homing and mobilization by enhancing or inhibiting CXCL12/CXCR4 signaling pathways and that the FL/Flt3 axis participates in trafficking of normal and transformed hematopoietic cells.