Intraepithelial T lymphocyte cells (IEL) are the first immune cells to respond to pathogens; they help maintain the integrity of the epithelial barrier. We studied the function of the mouse glycoprotein Signaling Lymphocyte Activation Molecule Family receptor (SLAMF) 4 (encoded by Slamf4) on the surface of CD8αβ αβ T-cell receptor (TCR)(+) IELs, and the roles of these cells in homeostasis of the small intestine in mice.SLAMF4(-) CD8(+) αβTCR(+) cells isolated from spleens of OT-I Rag1(-/-) mice were induced to express gut-homing receptors and transferred to C57BL/6J mice; levels of SLAMF4(+) cells were measured in small intestine tissues. After administration of anti-CD3 or antigen, with or without anti-SLAM4, to C57BL/6J and Slamf4(-/-) mice, CD8αβ αβTCR(+) IELs were collected; cytokine production and cytotoxicity were measured. Depletion of CX3CR1(+) phagocytes was assessed in mice by live-cell confocal imaging or by cytofluorometry; small intestine tissues were analyzed by histology and inflammation was quantified.Splenic CD8(+) αβTCR(+) cells began to express SLAMF4 only after migrating to the small intestine. Injection of C57BL/6J mice with anti-SLAMF4 and anti-CD3 increased levels of interleukin 10 and interferon gamma secretion by IEL, compared with injection of anti-CD3 only. Similarly, the number of granzyme B(+) cytotoxic CD8(+) αβTCR(+) IELs increased in Slamf4(-/-) mice after injection of anti-CD3 and anti-SLAMF4, administration of antigen, or injection of anti-CD3. Surprisingly, in vivo activation of CD8αβ(+) IELs with anti-CD3 or antigen caused transient depletion of CX3CR1(+) phagocytes, which was prolonged by co-injection with anti-SLAMF4 or in Slamf4(-/-) mice. Anti-CD3 aggravated inflammation in the small intestines of Slamf4(-/-) mice and Eat2a(-/-)Eat2b(-/-) mice, indicated by flattened villi and crypt hyperplasia.In mice, the intestinal environment induces SLAMF4 expression and localization to the surface of CD8(+) αβTCR(+) IELs. Signaling via SLAMF4 controls expansion of cytotoxic CD8αβ(+) IELs, which regulate the reversible depletion of lamina propria phagocytes and inflammation in the small intestine.