CD40 is a member of the TNFR family and upon interaction with its cognate ligand (CD40L), induces diverse biologic responses related to cell survival/growth. As altered CD40/CD40L interactions have been associated with neutropenia, we investigated the role of CD40/CD40L on human granulopoiesis using immunomagnetically sorted CD34(+), CD34(-)/CD33(+), and CD34(-)/CD33(-)/CD15(+) BM cells, which represent sequential stages of the granulocytic development, the KG-1 cells that constantly express CD34 and CD33, and LTBMCs that mimic the BM microenvironment. CD40 and CD40L were minimally expressed on CD34(+), CD34(-)/CD33(+), and CD34(-)/CD33(-)/CD15(+) cells, but CD40 was substantially induced in the presence of TNF-α. Cross-linking of CD40 in the above cell populations resulted in induction of apoptosis that was enhanced further in the presence of FasL. CD40 activation in primary as wells as in KG-1 cells resulted in Fas up-regulation, providing a mechanism for the CD40-mediated apoptosis. Addition of CD40L in clonogenic assays resulted in a significant decrease in the colony-forming capacity of BMMCs from patients with chronic neutropenia, presumably expressing high levels of CD40 in the progenitor cells, and this effect was reversed upon CD40 blockade. CD40 was constitutively expressed on LTBMC stromal cells and upon activation, resulted in an increase in G-CSF and GM-CSF production. These data show that CD40/CD40L interactions may promote granulopoiesis under steady-state conditions by inducing the stromal release of granulopoiesis-supporting cytokines, whereas under inflammatory conditions, they may affect the granulocytic progenitor/precursor cell survival by accelerating the Fas-mediated apoptosis.