Estrogen receptor (ER) ligands modulate hemopoiesis and immunity in the normal state, during autoimmunity, and after infection or trauma. Dendritic cells (DC) are critical for initiation of innate and adaptive immune responses. We demonstrate, using cytokine-driven culture models of DC differentiation, that 17-β-estradiol exerts opposing effects on differentiation mediated by GM-CSF and Flt3 ligand, the two cytokines that regulate DC differentiation in vivo. We also show that estradiol acts on the same highly purified Flt3+ myeloid progenitors (MP) to differentially regulate the DC differentiation in each model. In GM-CSF-supplemented cultures initiated from MP, physiological amounts of estradiol promoted differentiation of Langerhans-like DC. Conversely, in Flt3 ligand-supplemented cultures initiated from the same MP, estradiol inhibited cell survival in a dose-dependent manner, thereby decreasing the yield of plasmacytoid and conventional myeloid and lymphoid DC. Experiments with bone marrow cells from ER-deficient mice and the ER antagonist ICI182, 780 showed that estradiol acted primarily via ERα to regulate DC differentiation. Thus, depending on the cytokine environment, pathways of ER signaling and cytokine receptor signaling can differentially interact in the same Flt3+ MP to regulate DC development. Because the Flt3 ligand-mediated differentiation pathway is important during homeostasis, and GM-CSF-mediated pathways are increased by inflammation, our data suggest that endogenous or pharmacological ER ligands may differentially affect DC development during homeostasis and disease, with consequent effects on DC-mediated immunity.