The positive transcription elongation factor b (P-TEFb) stimulates transcriptional elongation by phosphorylating the carboxy-terminal domain of RNA polymerase II and antagonizing the effects of negative elongation factors. Not only is P-TEFb essential for transcription of the vast majority of cellular genes, but it is also a critical host cellular cofactor for the expression of the human immunodeficiency virus (HIV) type 1 genome. Given its important role in globally affecting transcription, P-TEFb's activity is dynamically controlled by both positive and negative regulators in order to achieve a functional equilibrium in sync with the overall transcriptional demand as well as the proliferative state of cells. Notably, this equilibrium can be shifted toward either the active or inactive state in response to diverse physiological stimuli that can ultimately affect the cellular decision between growth and differentiation. In this review, we examine the mechanisms by which the recently identified positive (the bromodomain protein Brd4) and negative (the noncoding 7SK small nuclear RNA and the HEXIM1 protein) regulators of P-TEFb affect the P-TEFb-dependent transcriptional elongation. We also discuss the consequences of perturbations of the dynamic associations of these regulators with P-TEFb in relation to the pathogenesis and progression of several major human diseases, such as cardiac hypertrophy, breast cancer, and HIV infection.