Systems biology has the potential to identify gene signatures associated with vaccine immunogenicity and protective efficacy. The main objective of this study was to identify optimal postvaccination time points for evaluating peripheral blood RNA expression profiles in relation to vaccine immunogenicity and potential efficacy in recipients of the candidate tuberculosis vaccine M72/AS01. In this phase II open-label study (NCT01669096; https://clinicaltrials.gov/), healthy Bacillus Calmette–Guérin-primed, HIV-negative adults were administered two doses (30 days apart) of M72/AS01. Twenty subjects completed the study and 18 subjects received two doses. Blood samples were collected pre-dose 1, pre-dose 2, and 1, 7, 10, 14, 17, and 30 days post-dose 2. RNA expression in whole blood (WB) and peripheral blood mononuclear cells (PBMCs) was quantified using microarray technology. Serum interferon-gamma responses and M72-specific CD4⁺ T cell responses to vaccination, and the observed safety profile were similar to previous trials. Two different approaches were utilized to analyze the RNA expression data. First, a kinetic analysis of RNA expression changes using blood transcription modules revealed early (1 day post-dose 2) activation of several pathways related to innate immune activation, both in WB and PBMC. Second, using a previously identified gene signature as a classifier, optimal postvaccination time points were identified. Since M72/AS01 efficacy remains to be established, a PBMC-derived gene signature associated with the protective efficacy of a similarly adjuvanted candidate malaria vaccine was used as a proxy for this purpose. This approach was based on the assumption that the AS01 adjuvant used in both studies could induce shared innate immune pathways. Subjects were classified as gene signature positive (GS⁺) or gene signature negative (GS⁻). Assignments of subjects to GS⁺ or GS⁻ groups were confirmed by significant differences in RNA expression of the gene signature genes in PBMCs at 14 days post-dose 2 relative to prevaccination and in WB samples at 7, 10, 14, and 17 days post-dose 2 relative to prevaccination. Hence, in comparison with a prevaccination, 7, 10, 14, and 17 days postvaccination appeared to be suitable time points for identifying potentially clinically relevant transcriptome responses to M72/AS01 in WB samples.