Fms-like tyrosine kinase 3 ligand (Flt3L) is a major cytokine that drives development of dendritic cells (DCs) under steady state, whereas GM-CSF becomes a prominent influence on differentiation during inflammation. The influence GM-CSF exerts on Flt3L-induced DC development has not been thoroughly examined. Here, we report that GM-CSF alters Flt3L-induced DC development. When BM cells were cultured with both Flt3L and GM-CSF, few CD8+ equivalent DCs or plasmacytoid DCs developed compared to cultures supplemented with Flt3L alone. The disappearance of these two cell subsets in GM-CSF + Flt3L culture was not a result of simple inhibition of their development, but a diversion of the original differentiation trajectory to form a new cell population. As a consequence, both DC progeny and their functions were altered. The effect of GM-CSF on DC subset development was confirmed in vivo. First, the CD8+ DC numbers were increased under GM-CSF deficiency (when either GM-CSF or its receptor was ablated). Second, this population was decreased under GM-CSF hyperexpression (by transgenesis or by Listeria infection). Our finding that GM-CSF dominantly changes the regulation of DC development in vitro and in vivo has important implications for inflammatory diseases or GM-CSF therapy.