Osteoblast cells mature from a mesenchymal stem cell pool to become cells capable of forming bone matrix and mineralizing this matrix. The goal of this study was to characterize temporal changes in the transcriptome across osteoblast maturation, starting with committed mesenchymal stem cell/ early pre-osteoblast stage through to mature osteoblasts capable of matrix mineralization. Methods: Enriched populations of pre-osteoblast-like cells were obtained from neonatal calvaria from B6xCAST mice expressing CFP under the control of the Col3.6 promoter. These cells were placed into culture for 4 days, removed from culture and subjected to FACS sorting based on the presence/absence of CFP expression. Cells expressing CFP were returned to culture, subjected to an osteoblast differentiation cocktail and RNA was collected at 2, 4, 6, 8, 10, 12, 14, 16 and 18 days post differentiation. Methods II: mRNA profiles for each time point were generated by next generation RNA sequencing, using an Illumina HiSeq 2000. Three technical replicates per sample were sequenced. Overall design: Gene expression in calvarial osteoblasts from neonatal B6xCAST-Col3.6 CFP mice at 9 time points post differentiation. File description: the R data files (.rda) provide outputs of the scripts in the mikelove/osteoblast-quant GitHub repo (July 2022, commit 01d96490), having run the fishpond package function importAllelicCounts() followed by minimal filtering. The `_counts.rda` files contain SummarizedExperiment objects with estimated count, TPM abundance, and effective length, but do not contain inferential replicates (bootstrap counts), although the transcript-level allelic counts object contains bootstrap mean and variances for every isoform, sample, and allele. The other two `.rda` files are summarized to gene level. The `_quant_dirs.tgz` files contain all the Salmon quantification data including bootstraps for the 9 time points. They are grouped into sets of three for convenience. The `CAST_EiJ.diploid.fa.gz` file provides the transcript sequences that were used for Salmon quantification. The `B6xCAST_discordant_global_AI.csv` file contains the same information as presented in Table S1 of Wu et al (2022). These are TSS-level results for 134 genes showing significant and discordant patterns within gene. The other 6 CSV files provide global and dynamic AI testing results at three levels of resolution: gene level, isoform level (txp), and TSS level where TSS within 50bp are combined into a single TSS-group. The significance cutoff is a q-value of 0.05. The code used for generating these results is provided in the GitHub repo: FennecFish/osteoblast-test.