Dendritic cell (DC) subsets can mediate diverse responses, but little is known about the Toll-like receptor (TLR) signalling pathways in different human DC subsets. Despite expressing many TLRs in common, we found that in vitro-derived Langerhans cells (LCs) and monocyte-derived DCs (moDCs) undergo differential signalling events following TLR stimulation. TLR-stimulated LCs did not secrete interleukin (IL)-12p70 and thus induced a T helper type 2 (Th2)-biased response. moDCs secrete high levels of IL-12p70 and induce a Th1 response. Stimulation of moDCs through TLR2 or TLR7/8 was able to induce phosphorylation of the mitogen-activated protein kinase (MAPK) extracellular-signal-regulated kinase (ERK). However, phosphorylated ERK was not induced in TLR-stimulated LCs, suggesting an ERK-independent method of Th2 cell induction. Inhibition of p38 MAPK suppressed moDC maturation, but was much less effective at inhibiting LC maturation. Phosphatidylinositol-3 kinase (PI3K) was also found to play a greater role in moDC survival compared with the LCs. Polymerase chain reaction (PCR) arrays to compare the expression of signalling molecules in LCs and moDCs identified differences in TLR recognition molecules and cytokine response genes, suggesting that differential functional responses are probably mediated at the post-transcriptional level. Thus we have described differences in LC and moDC responses to TLR stimulation, and have identified key differences in ERK phosphorylation and the involvement of MAPK and PI3K.