Gene functions in Trypanosoma brucei parasites are investigated by several reverse genetic approaches. One of the widely used tools is the tetracycline-inducible system, which allows controlled overexpression of a gene of interest or knockdown of gene expression by RNA interference. For studying multiple genes, it is desirable to be able to control their expression independently or together, as required. A tetracycline- and vanillic acid-based double-inducible expression system was previously described for procyclic-form trypanosomes. Here, we describe the adaptation of this double inducible system to bloodstream-form trypanosomes. We show that this system can be used for stringent, independent, and simultaneous regulation of the expression of two genes.