Keratinocytes (KCs) from healthy donors stimulated with type 2 cytokines are often used to experimentally study atopic dermatitis (AD) inflammatory responses. Owing to potential intrinsic alterations, it seems favorable to use KCs from patients with AD. KCs isolated from hair follicles offer a noninvasive approach to investigate AD-derived KCs. To evaluate whether such AD-derived KCs are suitable to mimic AD inflammatory responses, we compared hair follicle-derived KCs from healthy donors with those from patients with AD in a type 2 cytokine environment. Stimulation of AD-derived KCs with IL-4 and IL-13 induced higher expression changes of AD-associated markers than that of healthy KCs. The combination of IL-4 and IL-13 generally induced highest expression changes, but IL-13 alone also induced significant changes of AD-specific markers. Similar to the 2-dimensional cultures, IL-4/IL-13 stimulation of 3-dimensional skin models generated with AD-derived KCs modulated the expression of several AD-relevant factors. Whole-transcriptome analysis revealed that IL-4 and IL-13 acted similarly on these 3-dimensional skin models. Histologically, IL-13 alone and in combination with IL-4 increased epidermal spongiosis, a histological hallmark of AD skin. Taken together, our pilot study suggests that hair follicle-derived KCs from patients with AD represent a useful model system to study AD-related inflammation in a personalized in vitro model.