Folliculogenesis is a coordinated process, and the genes that regulate development are difficult to investigate in vivo. In vitro culture systems permit the assessment of individual follicles during development, thereby enabling gene expression patterns to be monitored during follicle development. Mouse multilayered secondary follicles (150–180 μm in diameter) were cultured in three-dimensional matrices of varying physical properties for up to 8 days. During this period of follicle growth in vitro, antrum formation and steroid production were monitored, and mRNA was isolated. The expression levels of genes (Star, Cyp11a1, Cyp17a1, Hsd3b1, Cyp19a1, Fshr, Lhcgr, Aqp7, Aqp8, Aqp9, and Hif1a) were measured and correlated to follicle developmental status. Follicles that developed an antrum and produced appropriate levels of estrogen and progesterone had unchanging expression of Star, Aqp7, Aqp8, and Hif1a and a 34-fold increase in Cyp19a1 expression at Day 8 of culture and had elevated Lhcgr at Days 6 and 8 of culture. Follicles that were healthy but did not form an antrum or produce appropriate levels of steroids, however, demonstrated increasing levels of Star, Aqp7, Aqp8, and Hif1a and a 15-fold increase in Cyp19a1 at Day 8 of culture, and Lhcgr levels were not elevated until Day 8 of culture. To our knowledge, this study provides the first temporal analysis of gene expression using individual culture in alginate hydrogels that correlates growth and steroidogenesis during follicle development and identifies expression patterns in healthy follicles and in developmentally disadvantaged follicles.