Fibroblast growth factor (FGF)-21, a structural relative of FGF23 that regulates phosphate homeostasis, is a regulator of insulin-independent glucose transport in adipocytes and plays a role in the regulation of body weight. It also regulates ketogenesis and adaptive responses to starvation. We report that in a reconstituted receptor activation assay system using BaF3 cells, which do not endogenously express any type of FGF receptor (FGFR) or heparan sulfate proteoglycan, FGF21 alone does not activate FGFRs and that Klotho is required for FGF21 to activate two specific FGFR subtypes: FGFR1c and FGFR3c. Co-expression of Klotho and FGFR1c on BaF3 cells enabled FGF21, but not FGF23, to activate receptor signaling. Conversely, co-expression of FGFR1c and Klotho, a protein related to Klotho, enabled FGF23 but not FGF21 to activate receptor signaling, indicating that expression of Klotho/Klotho confers target cell specificity on FGF21/FGF23. In all of these cases heparin enhanced the activation, but was not essential. In 3T3-L1 adipocytes, upregulation of glucose transporter expression by FGF21 was associated with expression of Klotho, which was absent in undifferentiated 3T3-L1 fibroblasts. It is thus suggested that Klotho expression is a crucial determinant of the FGF21 specificity of the target cells upon which it acts in an endocrine fashion.