We previously demonstrated that IL-10 alone does not stimulate growth and differentiation of human monocytes, but enhances those of monocytes stimulated with M-CSF. We studied here the effect of IL-10 on human monocytes stimulated with GM-CSF. Monocytes stimulated with GM-CSF alone survived and developed into macrophages. Monocytes cultured with GM-CSF plus IL-10, however, died through apoptosis. IL-10 decreased expression of bcl-2, bcl-x(L), and mcl-1- but not bax mRNA in monocytes stimulated with GM-CSF. IL-10 did not change the expression of mRNA of both GM-CSFR alpha-chain and beta-chain, but inhibited tyrosine phosphorylation of STAT5 and extracellular signal-regulated kinases 1 and 2 in the monocytes. The inhibitory effect of IL-10 was restricted to treatment 48 h after stimulation with GM-CSF. Addition of IL-10 after that time induced neither apoptosis nor a decrease in expression of bcl-2, bcl-x(L), and mcl-1 mRNA. IL-10, however, inhibited LPS-induced TNF-alpha production even in these cells, indicating that the cells still possessed responsiveness to IL-10. Monocytes pretreated for >48 h with GM-CSF became resistant to GM-CSF withdrawal, and the cells could survive without GM-CSF. These results indicate that IL-10 selectively inhibits GM-CSF-dependent monocyte survival by inhibiting the signaling events induced by GM-CSF, but the timing of addition of IL-10 is critical, and IL-10 had to be added within 48 h after stimulation with GM-CSF to achieve the inhibitory effect. These results taken together with our previous results indicate that IL-10 plays a pivotal role in monocyte survival and development into macrophages in concert with M-CSF and GM-CSF.