Background <p>Keratinocyte stem cells are essential for maintaining epidermal homeostasis and enabling efficient tissue repair. Regulation of their self-renewal and differentiation is critical, as its disruption can impair regeneration and drive pathological conditions such as chronic wounds and cancer. We previously identified the matricellular protein Mindin as a key regulator of keratinocyte stemness through its interaction with the αMβ2 (CD11b/CD18) integrin and subsequent activation of the transcription factor STAT3. However, the mechanism connecting Mindin and integrin at the cell surface to the intracellular activation of STAT3 remained undefined.</p> Methods <p>We employ biochemical and imaging analysis along with molecular dynamics simulations to dissect Mindin–integrin–STAT3 signalling in primary mouse keratinocytes. Stemness of epidermal keratinocytes are assessed using bulk RNA sequencing, quantitative PCR, and cell-based assays.</p> Results <p>Our work demonstrates that the F-Spondin domain of Mindin constitutes the minimal integrin-binding module required to initiate downstream signalling. F-Spondin binding to the integrin at the plasma membrane does not elicit the full activation state of the integrin. Instead, it promotes Src-kinase dependent endocytosis of the integrin receptor to the early endosomes. Analysis of integrin conformational dynamics reveals that the acidic environment of early endosomes is essential to achieve a signalling-competent state. This mechanism extends to pathological contexts, as we demonstrate a requirement for endocytosis in activating STAT3 signalling and preserving stem-like properties in a cancer stem cell model.</p> Conclusions <p>These findings highlight a previously unrecognized layer of spatial control in integrin signalling, confirming endosomal trafficking as a critical determinant of stem cell behaviour and offering new conceptual and therapeutic opportunities across regenerative biology and cancer.</p>