In addition to upregulating GM-CSF mRNA, AX-II increased RANKL mRNA expression dose-dependently in unfractionated human bone marrow mononuclear cells and modestly increased soluble RANKL in unfractionated human bone marrow mononuclear cell conditioned medium. However, AX-II markedly increased membrane-bound RANKL on human bone marrow stromal cells. Treatment of marrow stromal cells with AX-II activated MAP-kinase (ERKs) and PD 98059 abolished the effect but did not block the increase in GM-CSF. Interestingly, OPG, a natural decoy receptor for RANKL, or anti-GM-CSF partially inhibited OCL formation by AX-II in human bone marrow cells, and the combination of OPG and anti-GM-CSF completely blocked AX-II-induced OCL formation. These data show that AX-II stimulates both the proliferation and differentiation of OCL precursors through production of GM-CSF and RANKL respectively.