We describe two gene-knockout (KO) strategies in Trypanosoma brucei using Cre recombinase and loxP sites. Due to the limited number of selection markers for T. brucei, it has been difficult to generate a mutant with two genes knocked out and impractical to simultaneously knockout more than two genes, deterring detailed studies of important cellular mechanisms. The first KO strategy described can overcome the marker problem by allowing continuous re-use of drug-resistance markers. The same KO vector can be used to make a conditional KO system, when a gene of interest is essential for cell viability. As a gene of interest is removed from its original chromosomal locus by the induction of Cre recombinase, deletion is complete and instantaneous. This makes it easier to identify primary effects rather than having secondary effects obscuring phenotypic assessment, as is often the case with RNAi silencing.