Streptococcus pneumoniae (the pneumococcus) continues to be responsible for a high level of global morbidity and mortality resulting from pneumonia, bacteremia, meningitis, and otitis media. Here we have used a novel technique involving niche-specific, genome-wide in vivo transcriptomic analyses to identify genes upregulated in distinct niches during pathogenesis after intranasal infection of mice with serotype 4 or 6A pneumococci. The analyses yielded 28 common, significantly upregulated genes in the lungs relative to those in the nasopharynx and 25 significantly upregulated genes in the blood relative to those in the lungs in both strains, some of which were previously unrecognized. The role of five upregulated genes from either the lungs or the blood in pneumococcal pathogenesis and virulence was then evaluated by targeted mutagenesis. One of the mutants (ΔmalX) was significantly attenuated for virulence in the lungs, two (ΔaliA and ΔilvH) were significantly attenuated for virulence in the blood relative to the wild type, and two others (ΔcbiO and ΔpiuA) were completely avirulent in a mouse intranasal challenge model. We also show that the products of aliA, malX, and piuA are promising candidates for incorporation into multicomponent protein-based pneumococcal vaccines currently under development. Importantly, we suggest that this new approach is a viable complement to existing strategies for the discovery of genes critical to the distinct stages of invasive pneumococcal disease and potentially has broad application for novel protein antigen discovery in other pathogens such as S. pyogenes, Haemophilus influenzae type b, and Neisseria meningitidis.