The plasma membrane (PM) harbors small, liquid-ordered domains called “nanodomains,” which are primarily composed of sterols and sphingolipids. In plants, numerous immune proteins, including receptors, are accumulated in nanodomains; therefore, nanodomains play pivotal roles in plant immunity. However, the mechanisms underlying nanodomain-mediated plant immune regulation remain unknown. Recently, we generated a fluorescent probe, “GFP-D4L,” which allows for visualizing plant nanodomains by binding to sterols. Arabidopsis lines constitutively expressing GFP-D4L enable effortless observation of sterol-derived nanodomains. The present study clarified nanodomain dynamics during disease response using Arabidopsis GFP-D4L lines. While nanodomains were homogeneously distributed on PM under normal conditions, their distribution was heterogeneous following treatment with flg22, which induces Arabidopsis defense response. Specifically, flg22 is recognized by the pattern-recognition receptor FLS2 and the co-receptor BAK1 localized on PM, inducing a cellular defense signal. Under normal conditions, both FLS2–tagRFP and BAK1–tagRFP fused with GFP-D4L, indicating that both receptors are localized on nanodomains. However, following flg22 treatment, FLS2–tagRFP was not localized on nanodomains, whereas BAK1–tagRFP was retained on nanodomains. To examine the link between the distribution of nanodomains and receptors, we observed GFP-D4L distribution in fls2 mutants. In fls2 mutant, GFP-D4L showed heterogeneous distribution on PM even in the absence of flg22 treatment. Therefore, FLS2 may be involved in nanodomain distribution on PM. Additionally, we simultaneously treated GFP-D4L-expressing lines with flg22 and wortmannin, an endocytosis inhibitor, and observed homogeneous distribution of GFP-D4L on PM. Therefore, the heterogeneous distribution of nanodomains under flg22 treatment indeed depends on FLS2 endocytosis.