Efficient pluripotent stem cell guidance protocols for the production of human posterior cranial placodes such as the otic placode that gives rise to the inner ear do not exist. Here we use a systematic approach including defined monolayer culture, signaling modulation, and single-cell gene expression analysis to delineate a developmental trajectory for human otic lineage specification in vitro. We found that modulation of bone morphogenetic protein (BMP) and WNT signaling combined with FGF and retinoic acid treatments over the course of 18 days generates cell populations that develop chronological expression of marker genes of non-neural ectoderm, preplacodal ectoderm, and early otic lineage. Gene expression along this differentiation path is distinct from other lineages such as endoderm, mesendoderm, and neural ectoderm. Single-cell analysis exposed the heterogeneity of differentiating cells and allowed discrimination of non-neural ectoderm and otic lineage cells from off-target populations. Pseudotemporal ordering of human embryonic stem cell and induced pluripotent stem cell-derived single-cell gene expression profiles revealed an initially synchronous guidance toward non-neural ectoderm, followed by comparatively asynchronous occurrences of preplacodal and otic marker genes. Positive correlation of marker gene expression between both cell lines and resemblance to mouse embryonic day 10.5 otocyst cells implied reasonable robustness of the guidance protocol. Singlecell trajectory analysis further revealed that otic progenitor cell types are induced in monolayer cultures, but further development appears impeded, likely because of lack of a lineage-stabilizing microenvironment. Our results provide a framework for future exploration of stabilizing microenvironments for efficient differentiation of stem cell-generated human otic cell types.posterior placode | ectoderm | gene expression analysis | inner ear | pluripotent stem cells V ertebrate cranial placodes arise from a region of non-neural ectoderm (NNE) lateral to the rostral neural crest progenitor region and the neural plate (reviewed in refs. 1 and 2). In humans, the cranial placodes form during the first month of gestation, restricting our insight to experiments using pluripotent stem cell-based models. Generation of human NNE and derivation of anterior placodal cells (i.e., pituitary, lens, and trigeminal neurons) from human embryonic stem cells (hESCs) has previously been achieved (3, 4). Production of posterior human placodal fates such as the otic placode and epibranchial ganglia neurons remains elusive, despite indications that immature sensory hair cell-like cells might arise in hESC-derived aggregates via manipulation of TGFβ, WNT, and FGF signaling, albeit with low efficiency (5-7).We used adherently grown hESCs and human induced pluripotent stem cells (iPSCs) to systematically test conditions for stepwise induction of NNE to posterior placode fates; specifically, the otic lineage. A challenge of in vitro guidance is the transient state of presump...