Lymphocyte homing is mediated by a specific interaction between L-selectin and its sulfated glycoprotein ligands expressed on high endothelial venules (HEVs) in lymph nodes. To examine the significance of sulfation of L-selectin ligands, gene targeting mice deficient in both N-acetylglucosamine-6-O-sulfotransferase (GlcNAc6ST)-1 and GlcNAc6ST-2 (HEC-GlcNAc6ST/ LSST) have been generated. In the double-knockout mice, binding of MECA-79 antibody to lymph node HEV was completely abolished, indicating that extended core 1 O-glycans containing GlcNAc-6-O-sulfate is completely diminished in those mice. Furthermore, the mutant mice showed approximately 75% less lymphocyte homing to the peripheral lymph nodes (PLNs) and significantly less contact hypersensitivity response than wild-type mice, demonstrating that GlcNAc6ST-1 and GlcNAc6ST-2 play a major role in L-selectin ligand biosynthesis in HEVs. In this chapter, the detailed protocols that have been used for the functional assays of these sulfotransferase double-knockout mice are described.