Osteogenic differentiation is essential for bone development and metabolism, but the underlying gene regulatory networks have not been well investigated. We differentiated mesenchymal stem cells, derived from 20 human induced pluripotent stem cell lines, into preosteoblasts and osteoblasts, and performed systematic RNA-seq analyses of 60 samples for differential gene expression. We noted a highly significant correlation in expression patterns and genomic proximity among transcription factor (TF) and long noncoding RNA (lncRNA) genes. We identified TF-TF regulatory networks, regulatory roles of lncRNAs on their neighboring coding genes for TFs and splicing factors, and differential splicing of TF, lncRNA, and splicing factor genes. TF-TF regulatory and gene co-expression network analyses suggested an inhibitory role of TF KLF16 in osteogenic differentiation. We demonstrate that in vitro overexpression of human KLF16 inhibits osteogenic differentiation and mineralization, and in vivo Klf16 +/- mice exhibit increased bone mineral density, trabecular number, and cortical bone area. Thus, our model system highlights the regulatory complexity of osteogenic differentiation and identifies novel osteogenic genes.