Stromal cell-derived factor-1α (SDF-1α) is a strong migratory stimulant for hematopoietic stem and progenitor cells (HSPCs). The hematopoietic cytokines thrombopoietin (TPO), Flt3-ligand (FL), stem cell factor (SCF) and interleukin 11 (IL-11) are able to stimulate amplification of primitive murine hematopoietic stem cells (HSCs) in vitro. The effects of these cytokines on SDF-1α-induced migratory activity of murine Lin^-c-kit⁺ HSPC were analyzed by cultivation of these cells in the presence of 12 combinations of FL, TPO, SCF and IL-11. Migratory activity was measured in a three-dimensional collagen matrix using time-lapse video microscopy. Each cytokine combination had a distinct effect on SDF-1α-stimulated migratory activity. For instance, FL- and SCF-cultivated cells showed a high migratory SDF-1α response, while cells cultivated with SCF, TPO and IL-11 did not react to SDF-1α stimulation with an elevated migration rate. Our data indicate that the differences in the migratory SDF-1α response are not related to different CXCR4 expression levels, but rather to the differential engagement of the CXCR4-dependent MAPK^p42/44 and PI3K signal transduction pathways. This indicates that hematopoietic cytokines can have a significant impact on SDF-1α-stimulated migratory activity and the underlying intracellular signaling processes in cultivated HSPCs.