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 Glut1 and/or Glut3, in either the incipient pre-skeletal mesenchyme with Prx1Cre or in chondrocytes with Col2Cre, disrupted interzone formation dose-dependently. In contrast, deletion of Glut1/3 in established interzone cells with Gdf5Cre did not have similar severe disruption of joint development. scRNA-seq revealed that Glut1/3 deletion by Prx1Cre impeded Tgfβ signaling in the developing interzone cells. Direct elimination of Tgfβ signaling with Prx1Cre partially phenocopied the deletion of Glut1/3 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.