The impact of CD44 inhibition on the expression of proteins involved in the Wnt/β-catenin signaling pathway, as well as on other CSC markers like Oct4 and Klf4, was examined. Inhibiting CD44 led to reduced levels of β-catenin and phosphorylation of GSK3β (Ser 9), alongside decreased expression of other CSC markers. Treatment with 1 mM THIQ resulted in a significant reduction in CD44 expression in both cisplatin-resistant HNSCC cells and their parental counterparts. Additionally, a flow cytometric analysis revealed that 1 mM THIQ treatment decreased the percentage of CD44-positive cells. Here, POU5F1 is linked to head and neck squamous cell carcinoma.