Numerous mechanisms underlie chronic graft-versus-host disease (cGVHD), including skewing of Th1/Th2 cytokine expression. There are biological differences between early-onset and late-onset cGVHD. To test whether different Th1/Th2 cytokines are associated with early- or late-onset cGVHD, peripheral blood was collected from 63 children enrolled on the Children's Oncology Group Phase III trial ASCT0031 evaluating hydroxychloroquine therapy for newly diagnosed extensive cGVHD. mRNA expression of interferon (IFN)-γ and interleukin (IL)-2, -4, and -10 from stimulated peripheral blood mononuclear cells was evaluated by quantitative polymerase chain reaction. Early-onset cGVHD (n = 33) was characterized by decreased expression of IFN-γ and IL-2 mRNA after nonspecific phorbol 12-myristate 13-acetate–ionomycin stimulation. In contrast, late-onset cGVHD (n = 11) was characterized by decreased expression of IL-4 and IL-2 mRNA after anti-CD3 stimulation of T cells. Receiver-operating characteristic curve analysis revealed that IFN-γ expression was correlated with the absence of early cGVHD (area under the curve [AUC] = 0.77) and that IL-4 (AUC = 0.89) and IL-2 (AUC = 0.84) expression was correlated with the absence of late cGVHD. There was no correlation between cytokine expression and a specific immune cell subset. Increased expression of Foxp3 mRNA was seen in early-onset cGVHD and late controls. The different time-dependent cytokine profiles in patients with newly diagnosed cGVHD suggests that the mechanisms underlying cGVHD are temporally regulated. Although larger validation studies are needed, our data suggest that cytokine profiles have a potential use as biomarkers for the diagnosis of cGVHD.