We have previously reported that human umbilical cord blood-derived stromal cells (hUCBDSCs) are able to enhance the expansion of CFU-Meg in vitro, particularly promote the megakaryocytic lineage recovery, and effectively protect the survival of irradiated mice. In this study, we demonstrated that hUCBDSCs secreted SDF-1 to stimulate PECAM-1 expression in HEL cells (MK cell line), and consequently promoted the proliferation and migration of HEL cells. On the other hand, SDF-1 knock down in hUCBDSCs or PECAM-1 knock down in HEL cells diminished or abrogated the above effect. In addition, SDF-1/PECAM-1 probably activated PI3K/Akt and MAPK/ERK1/2 pathways. This report for the first time defines a SDF-1/PECAM-1 signaling pathway in the proliferation and migration of MKs, which provides supportive evidence for the clinical applications of hUCBDSCs in the treatment of megakaryocytic injury.