Plant formation from in vitro-cultivated microspores involves a complex network of internal and environmental factors. Haploids/doubled haploids (DHs) derived from in vitro-cultured microspores are widely used in plant breeding and genetic engineering. However, the mechanism underlying the developmental switch from regular pollen maturation towards microspore-derived plant regeneration remains poorly defined. Here, RNA-sequencing was employed to elucidate the transcriptional landscapes of four early stages of microspore embryogenesis (ME) in barley cultivars Golden Promise and Igri, which exhibit contrasting responsiveness to microspore-derived plant formation. Our experiments revealed fundamental regulatory networks, specific groups of genes, and transcription factor (TF) families potentially regulating the developmental switch. We identified a set of candidate genes crucial for genotype-dependent responsiveness/recalcitrance to ME. Our high-resolution temporal transcriptome atlas provides an important resource for future functional studies on the genetic control of microspore developmental transition.