Transcription by RNA polymerase II is regulated, in part, by the positive transcription elongation factor b (P-TEFb), which promotes transition from abortive to productive elongation. The Drosophila P-TEFb complex is composed of the Cdk9 kinase and a cyclin partner, CyclinT or CyclinK. To investigate the physiological role of P-TEFb, we generated transgenic flies allowing the conditional expression of wild-type or mutant Cdk9, alone or together with CyclinT or CyclinK. We found that the two P-TEFb complexes have similar binding pattern on chromosomes and are recruited to transcriptionally active loci. By expressing a dominant-negative form of Cdk9 in specific tissues, we showed that P-TEFb function is required for endoreplication of larval tissues, for proper differentiation of imaginal discs and for oogenesis. We demonstrated that the two cyclin subunits have non-redundant activities in vivo, and that P-TEFb containing CyclinT, but not CyclinK, activates transcription when tethered to promoter.