hESCs were cultured in differentiation conditions for 14 (upper panel) or 21 (lower panel) days in control, nicotine-supplemented, or tobacco smoke-exposed media, and expression of markers for embryonic ectoderm (Ecto), mesoderm (Meso) and endoderm (Endo) was evaluated by quantitative real-time PCR. Data shown are mean±SD (N=4). Persistent expression of the early mesodermal marker, Brachyury, and ectodermal marker, βIII tubulin, was seen despite culture under differentiation conditions at both time points. In addition, persistently elevated expression of markers for all three germ layers was observed at 21 days, suggesting a widespread delay in differentiation. In some cases, the changes in expression were similar with both tobacco smoke and nicotine treatment (i.e., nestin, Nkx2-5, α-fetoprotein), suggesting these effects may be predominantly due to nicotine, while for other markers, the effects were greater for tobacco smoke (i.e., βIII tubulin, Brachyury, Tal1, FoxA2), consistent with additive effects from other smoke components.