The initial interzone cells for synovial joints originate from chondrocytes, but such critical transition is minimally understood. With single-cell RNA sequencing (scRNA-seq) of murine embryonic knee joint primordia, we discovered that heightened expression of glycolysis genes characterized developing interzone cells when compared to flanking chondrocytes. Conditional deletion of the glucose transporters and/or , in either the incipient pre-skeletal mesenchyme with or in chondrocytes with , disrupted interzone formation dose-dependently. In contrast, deletion of / in established interzone cells with did not have similar severe disruption of joint development. scRNA-seq revealed that deletion by impeded Tgfβ signaling in the developing interzone cells. Direct elimination of Tgfβ signaling with partially phenocopied the deletion of in impairing interzone formation. Tgfβ stimulated glycolysis in chondrocytes via activation of mTOR and Hif1α in vitro. The data support that the essential conversion of chondrocytes to interzone cells requires a transient elevation of glycolysis partly dependent on Tgfβ signaling.