We have cultured Plasmodium falciparum directly from the blood of infected individuals to examine patterns of mature-stage gene expression in patient isolates. Analysis of the transcriptome of P. falciparum is complicated by the highly periodic nature of gene expression because small variations in the stage of parasite development between samples can lead to an apparent difference in gene expression values. To address this issue, we have developed statistical likelihood-based methods to estimate cell cycle progression and commitment to asexual or sexual development lineages in our samples based on microscopy and gene expression patterns. In cases subsequently matched for temporal development, we find that transcriptional patterns in ex vivo culture display little variation across patients with diverse clinical profiles and closely resemble transcriptional profiles that occur in vitro. These statistical methods, available to the research community, assist in the design and interpretation of P. falciparum expression profiling experiments where it is difficult to separate true differential expression from cell-cycle dependent expression. We reanalyze an existing dataset of in vivo patient expression profiles and conclude that previously observed discrete variation is consistent with the commitment of a varying proportion of the parasite population to the sexual development lineage.