Trypanosoma brucei is a protozoan parasite responsible for health and economic burden in some regions of sub-Saharan Africa. In T. brucei the mitochondrial genome is housed within a nucleoid termed the kinetoplast. The kinetoplast is physically connected to a cytoplasmic basal body (microtubule-organizing center for the flagellum). Duplication of both the kinetoplast and basal body are coordinated with trypanosome S-phase. Regulatory pathways which promote S-phase entry and control duplication of the kinetoplast and basal body are poorly understood in T. brucei. Here we describe a small molecule kinase inhibitor, AEE788, which inhibits duplication of the kinetoplast and basal body, and prevents DNA synthesis; effectively blocking trypanosome entry into S-phase. We developed an AEE788 block-and-release protocol for enriching bloodstream T. brucei in G1. Thus, for the first time we experimentally documented the kinetics of DNA synthesis (in the kinetoplast and nucleus), basal body duplication, kinetoplast division, and mitosis during trypanosome division, establishing AEE788 as a useful chemical tool for the study of trypanosome biology. A second study in this work demonstrates that reduced levels of a trypanosome casein kinase 1, TbCK1.2, caused amplification of basal bodies, while increased TbCK1.2 levels inhibited duplication of the organelle. Further, we detected TbCK1.2 at basal bodies, and demonstrated that phosphorylation of basal body proteins was altered after knockdown of the kinase. Interestingly, knockdown of TbCK1.2 inhibited kinetoplast division without preventing kinetoplast DNA (kDNA) replication, basal body duplication/separation, or flagellum biogenesis. These data are at odds with current dogma which describes basal body separation as the cause of kinetoplast division. Accordingly, we hypothesize that a regulatory pathway, dependent on TbCK1.2, is required to promote decatenation of the interlocked kDNA network. Taking into account our work, and other published data, we propose that proteins required for kinetoplast division (kinetoplast division factors) direct decatenation of the kDNA network to prevent asymmetric division. Collectively this work: i) identifies AEE788 as a chemical tool to reversibly enrich pre-S-phase bloodstream T. brucei, ii) demonstrates the role of TbCK1.2 in controlling basal body copy number, and iii) offers a new perspective on the regulatory pathways which are required for kinetoplast division.