Microenvironment molecular cues direct T helper (Th) cell differentiation; however, Th17 fate determination is still imprecisely understood in humans. To assess the role of prostaglandin E₂ (PGE₂) in Th expansion, we activated peripheral blood mononuclear cells by CD3 cross-linking. In the presence of exogenous PGE₂, peripheral blood mononuclear cells produced higher interleukin-17 (IL-17), C-C chemokine ligand 20 (CCL20)/macrophage inflammatory protein 3α (MIP-3α), CXC chemokine ligand 8 (CXCL8)/IL-8, and lower interferon-γ and IL-22 levels than in control cultures. Exogenous PGE₂ and IL-23 synergized in inducing IL-17, whereas indomethacin and IL-23 blockade drastically reduced IL-17 but not interferon-γ production. Furthermore, IL-1 but not tumor necrosis factor was absolutely required for IL-17 production. PGE₂ doubled the frequency of CD4⁺ T cells producing IL-17 and within the CD4⁺ subset enhanced C-C chemokine receptor 6 (CCR6) and CCR4 while decreasing CXC chemokine receptor 3 (CXCR3) expression. Furthermore, in CD4⁺ T-cell lines, the production of IL-17 segregated with the CCR6⁺ subset. In the presence of CCR6⁺ compared with CXCR3⁺ Th cells, monocytes/macrophages produced much higher levels of matrix metalloproteinase-1, -3, and -9 but similar levels of CXCL10 and IL-1β. These results identify PGE₂ and IL-23 as participating in the expansion of CD4⁺ T cells endowed with high IL-17 production capacity, which in turn favors monocyte production of mediators important for host defense and tissue destruction.